Protein delivery PharmDr. Veronika Mikušová, PhD.

Protein structure

• primary structure: amino acid chaines in well-defined 3D structure

• secondary structure: α helices, β sheets

• tertiary structure: proper positioning of the different subunits relativer to each other

• quarternary structure: individual protein molecules interact to build a larger well-defined structure ex: haemoglobin

• Formation and stability of sec., tert. and quarternary are based on relatively weak physical interactions:

▫ Electrostatic interactions

▫ Hydrogen bounding

▫ Van der Waals forces

▫ Hydrophobic interactions

▫ Not on covalent binding

which are relatively weak = protein structure can be rather easily changed (loss of pharmacological properties)

• Amino acid chaines can be modified by covalently attaching non-amino acid sections:

▫ sugars = glycoproteins

▫ phosphate groups

▫ sulphate groups

• These groups may be essential for the pharmacological effect of the protein

Proteins and GIT

• Pharmaceutical protein molecules are large • The GIT muconasal surface is a large interface that is

protected by a monolayer of epithelial cells, connected by tight junctions - it acts as a physical and chemical barrier towards the absorption of proteins and peptides

• Diffusional transport through epithelial barriers (GIT) is slow unless specific transporter molecules are available

• Conditions in GIT are extremely hostile to this proteins = enzymatic degradation is fast

• => large majority of pharmaceutical protein is therefore delivered via parenteral route

Possible mechanisms of proteins

absorption in GIT:

• Passive transport: simple or facilitated diffusion,

along concentration gradient, independent of chemical energy (but low lipophilicity and large Mr limit this process, beneficial for di/tripeptides)

• Active transport: against concentration gradient

• Endocytosis: energy consuming process – engulfment of the molecules for absorption

• Transcellular pathway: across the cells 1. by passive diffusion: depends on physico-chemical

properties of the molecule: size, charge and lipophilicity

2. by a specific carrier: peptide and amino acid transporters transport molecule from lumen into the cell – substrate specificity

• But all compounds absorbed through transcellular pathway are substrate for p-glycoprotein mediated efflux which transports the molecules from inside the cell back into the intestinal lumen

• The lipophilicity of a molecule is one of the main factors that governs transport via the transcellular route as molecules need to pass through the lipid bilayer of the intestinal membrane - a significant challenge

3. Transcytosis, Receptor-mediated endocytosis

• The transepithelial transport of macromolecules by intestinal epithelial cells occurs to a limited extent through absorption into lymphatic circulation via M-cells of Peyer's patches – minimal degradation of proteins - promising mechanism

• Paracellular pathway: transport of peptides across the aqueous channels in the cell junctions

• Not ideal for macromolecules (restricted to relatively small hydrophilic molecules)

• The addition of a novel functionality to the protein or the use of novel delivery systems – may enable optimal absorption

• Proteolytic enzymes: present at various regions along the GIT (trypsin, chymotrypsin, exopeptidases, endopeptidases etc.)

• Acidic nature of gastric medium – denaturation and degradation of protein

• Hydrolytic, irreversible cleavage of proteins into AA and small absorbable oligopeptides

• conserving the integrity of these large molecules is essential to:

▫ ensure an optimal therapeutic effect

▫ and to minimize effects such as the induction of unwanted immune responses (immune response may neutralize therapeutic activity in chronic dosing schedules and cause serious side-effects)

• Many functional groups are available for chemical degradation (and preferred 3D structure is irrevesibly disturbed) by for example:

▫ Heat

▫ pH changes

▫ Changes in ionic strenght

• Preferred shelf life for pharmaceutical produc is at minimum 2 years

• But most protein degrade too fast when formulated as aqueous solutions even when kept in the refrigerator

• Therefore they have to be stored in in a dry form and be reconstituted before administration

• They are usually dried by freeze-drying and the choice of proper excipients during this step (lyoprotectants) is extremely important

Sources of pharmaceutical proteins

• Most proteins used in therapy are produced by: ▫ recombinant DNA technology ▫ or hybridoma technology

• They are known as biotechnology products or bioitech products

• Ex: ▫ Human isulin ▫ Erythropoietin ▫ Monoclonal antibodies ▫ Cytokines ▫ Interferons

Production

• They are all produced in cell cultures by procaryotic or eucaryotic cells (E.coli, mamalian cells as Chinese hamster ovary cells or transgenic animals and genetically modified plants)

• Not all are exactly identical with endogenous product • Clinical trials has proved their efficacy and safety • Isolation of the expressend protein from the culture

medium is a multistep process consisting of several different steps (chromatography and filtration)

• For every protein a tailor made purification protocol has to be developed to remove impurities while ensuring integrity

• Majority of protein drug are biotech-derived molecules

• But there are still proteins of major therapeutic importance isolated from blood from humans or animals ▫ Albumin ▫ Blood clotting factors (factor VIII from the blood of

human volunteers) ▫ Antisera from patients or animals (horses, sheeps)

• Special purification protocols have to be developed, with particular emphasis on reduction of viral contamination

Formulation of pharmaceutical

proteins for parenteral administration

Physical instability • Depends on environmental conditions • Examples:

▫ Elevated temperature – denaturation of proteins in aqueous solutions

▫ Low temperature – may induce destabilization ▫ Adsorption of the protein monomer on the walls of the

container – protein aggregation ▫ Shaking, exposure to shear forces – protein

aggregation (hydrophobic parts of the molecule are exposed to hydrophobic interface air/water –> unfolding of the protein -> aggregation)

Chemical instability

• Full prevention of all chemical degradation reactions is difficult (because of many AA involved)

• Formulator should consider which chemical degradation pathways are relevant

• At neutral pH peptide bonds between AA are stable, only Asn-Gly and Asn-Pro are relatively labile

• Pathways of degradation of protein: ▫ Fragmentation ▫ Isomerisation ▫ Deamidation ▫ Oxidation ▫ Disulphide scrambling ▫ Oligomerization ▫ Aggregation ▫ Cross-linking ▫ Denaturation

Deamidation

• Rather common degradation in water

• Asn and Gln can be deamidated

• Kinetics depends on pH and neighbouring AA

→

Glu Gln

→

Oxidation • Met, Cys, His, Try, and Tyr are sensible • Oxidative milieu may also cause free Cys units to form disulphide

bridges or disulphide bind scrambling ▫ oxidation of Met to sulfoxide can be associated with the loss of

biological activity for many peptide hormones (i.e. corticotropine)

▫ oxidation can be iniciated by metal ions, visible light – photoxidation

▫ pH independent, may increase at low temperature (better solubility of O2 in water)

▫ the most important controling parameter: the degree of solvent accessibility to the side chains of proteins (burried side chains oxidize very slowly)

Isomerization, racemization

• Naturally occuring L-forms turns to D-forms, it changes activity of the protein

• Slow process occuring in vivo

Proteolysis • acidic hydrolysis of

peptide bonds of Asp

• hydrolysis can take place at either N-terminal and/or C-terminal peptide bond

Incorrect disulfide formation

• sulfhydryl groups and disulfide bonds are an important factor affecting the properties of proteins

• the interchange of disulfide bonds can result in incorrect pairings, leading to an altered 3D structure

Denaturation (the loss of the globular or 3D structure)

• thermal denaturation (often depends on pH; for lysozyme 42°C/pH=2, 65°C/pH=6.5)

• cold denaturation (proceeds often below -20°C) • chemical denaturation (urea, guanidinium

hydrochlorid) • pressure-induced denaturation (pressures 2000 –

4000 bar, fully reversible) • denaturation in the solid state (needs higher

temperatures, often above 150°C, irreversible)

Aggregation (the formation of soluble aggregates – association of monomers)

• aggregates can increase the likelihood of adverse immunogenic effects during therapy

• the biological function of the molecule can be compromised in non-native aggregates, thus reducing its efficacy

• aggregated protein can make a solution appear turbid or physically separate from the solution

Precipitation (the macroscopic event where the protein can be seen coming out of solution)

• it is often result of aggregation, when aggregates are so large that they can no longer remain soluble – irreversible process

• precipitation by salting-out – reversible process Adsorption (the adhesion of proteins to surfaces)

• adsorption changes the physical state of the protein • subsequent damage of protein can occur upon

interfacial stress • adsorption at air – water or ice – water inteface

• Degradation of protein may be also caused by the incorrect choice of excipients:

• Example: sugars are often used as excipients but reducing sugars (as lactose) can react with free primary amino groups of the protein via Maillard reaction (even in dry state) and form brownish reaction products = reducing sugars are excluded from formulation containing proteins

Excipients used Excipients used in parenteral dosage forms and their functions

Excipient Function Examples

Solubility-enhancing substances

Increase solubility of proteins

Amino acids, detergents

Antiadsorbent/aggregation blockers

Reduction of adsorption and aggregation prevention

Albumin, detergents

Buffer components Stabilizing pH Phosphate, citrate

Preservatives Growth inhibition in vials for multiple dosing

Phenol, benzylalcohol

antioxidants Prevent oxidation Ascorbic acid, sulphites, Cys

Stabilizers during storage (lyoprotectants)

Preservation of integrity while in dry form

Sugars

Osmotic compounds Ensure isotonicity Sugars, NaCl

Solubility enhancers

• Not all excipients are always needed: many pharmaceutical proteins are sufficiently soluble in water (highly glycosylated molecules) and no solubility enhancers are necessary

• If solubility enhancer is necessary, selection of proper pH conditions should first be considered

• Protein solubility depends on its net charge

• Uncharged proteins molecules (at the pH of their isoelectric point) have the lowest solubility on water – choose the pH condition away from their IEP

Antiadsorbent/aggregation blockers

• Detergents such as Polysorbate 20 and 80 or sodium dodecyl sulphate prevent the adsorption of protein to interfaces (air/water and container/water) and thereby interface-induced protein unfolding

• Human serum albumin has strong tendency to absorb to interface = antiaggregation agent (its use is discouraged – risk of transmitting BSE agents)

Antioxidants

• Oxidation reaction are catalysed by heavy metals

• Chelating agents are use to reduce oxidation damage through binding of the ions

• Not used if the metal ion is necessary as an integral part of the protein structure (Zn ions in insulin and Fe ions in haemoglobin) then antioxidants such as sulphites may be added

Preservatives

• In case of vials for multiple dosing, preservatives have to be included in the formulation

• Usually benzylalcohol and phenol

Stabilizers during storage

(lyoprotectants) • Buffered aqueous protein solutions may be stable

for 2 years under refrigerator conditions (some monoclonal antibodies)

• But often formulation has to be freeze dried in vials to avoid degradation

• Freeze drying: water is removed by sublimation • 3 phases:

1. freezing of the solution to -35 to -40 C 2. sublimation at - 35 C and at very low atmosphere

pressure to remove all frozen water 3. drying stage to remove most residual water (low

pressure, temperature can rise up to about 20 C)

• Lyoprotectant (e.g. sugar) is necessary to stablize the product as the removal of water may irreversibly affect the protein structure

• + they form readily reconstituable porous cakes

• Mechanisms of action of non-reducing sugars (not fully understood)

Microbiological requirements

• Pharmaceutical proteins are administered via parenteral route = sterile product

• PP cannot be sterilized by autoclaving, gas sterilization or ionizing radiation (cause damage of molecule) => sterilization of the endproduct is not possible

• => aseptic manufacturing is the only option: ▫ All utensils and components must be presterilized (by heat

sterilization, ionizing radiation or membrane filtration)

▫ Protein product are manufactured under aseptic conditions (air filtration through HEPA filters)

▫ Product is filled into the containers through sterile filters (0,22 µm pores) before capping or freeze drying/capping

• Protein product (PP) contamination: Viruses: ▫ by the use of contaminated culture media ▫ via infected (mammalian) production cells

• Viral decontamination steps are important in the purification and manufacturing protocols: ▫ Virus removal: Filtration Precipitation Chromatography

▫ And/or viral inactivation (problem: narrow window between succesful viral inactivation and preservation of the integrity of PP structure): Heat treatment (pasteurization) Radiation Crosslinking agents (e.g. β-propiolactone) none of them guarantees complete virus removal, often

several different decontamination steps are introduced in series

• Pyrogens: gram-negative host cell (e.g. E.coli) used as production cells contain large amounts od endotoxins in their membranes

• Endotoxines: heat stable, amphipatic, negatively charged lipopolysacchaides, potent pyrogens

• Pyrogen removal: for ex: anion-exchange chromatography

Analytical techniques to characterize

proteins • Total structure of protein is responsible for the

pharmacodynamic (e.g. receptor interaction) and pharmacokinetic (e.g. clearance, targeting) effects

• Not possible to define its structure with same precision as small, low molecular weight molecules

• Therefore a set of pharmacological, immunological, spectroscopic, electrophoretic and chromatographic approaches is used to characterize protein as closely as possible

• None of these tests tells the whole story, together they tell more but it is seldom possible to obtain a full description of the drug, including a detailed impurity profile

In vivo tests, use of test animals

• Information obtained: pharmacological effect

• Example: pharmacopoeial test for insulin:

▫ Lowering of the blood glucose level in rabbits upon injection of the insulin product to be tested

• these tests do not have the sensitivity:

▫ to identify small changes in molecular structure

▫ or detect early degradation products

▫ they do not provide information on the presence of product immunogenicity

In vitro tests (sensitive cells)

• Information obtained: Functional test – functional activity of the molecule

• Example: cytokine activity assessment

• Test do not inform about pharmacokinetic behaviour of the molecule or immunogenicity

Immunological tests

ELISA (enzyme-linked immunosorbent assay)

RIA (radioimmunoassay)

• Determination of the interaction of a monoclonal antibody with one epitope region on the protein

• The rest of the molecule is not probed

Analytical approaches

Spectroscopic • UV spectroscopy • Fluorimetry • CD spectroscopy (circular dichroism) • Infrared spectroscopy • Mass spectrometry as MALDI-TOF MS (matrix-

assisted laser desorption ionization time of flight mass spectroscopy), HPLC-electrospray ionization-induced MS (detailed information on amino acid sequence and glycosylation patterns)

• Information obtained: secondary/tertiary structure

Electrophoretic approaches

• SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis) (molecular weight)

• IEF (isoelectric focusing) (isoelectric point IEP)

• Powerful tools to asses product purity and to provide molecular weight and IEP information regarding the protein

HPLC (High performance liquid chromatography)

• GP (gel permeation) (basic molecular size –Mr + powerfull technique to monitor aggregate formation)

• HI (hydrophobic interaction) (hydrophobic interactions)

• Affinity chromatography (Interaction with specific ligand)

• IEC (ion-exchange chromatography) (separates on the basis of subtle variations in protein charge patterns, is used to detect oxidation (e.g. methionine) and deamidated (converted Gln and Asn products)

• RP (reverse phase) • Chromatographic techniques: impurities and

degradation products can be picked up at early stage

Routes of administration of PP

• Parenteral

• Oral

• Pulmonary

• Transdermal

• Rectal

• Ophtalmic

• Buccal

A. Parenteral protein delivery

• Intravenose: clearance from the blood

compartment can be ▫ fast, with half-life of minutes (ex: tissue plasminogen

activator (tPA)) ▫ slow, with half-life of several days (ex: human

monoclonal antibodies) • Subcutaneous, intramuscular: • More patient friendly • Protein is not instantaneously drained to the blood

compartment – s.c.passage of the protein through endothelial barrier lining the local capillaries at the site of injection is size dependent

• If the protein is too large – it enters lymphatic system and is transported via the lymph into the blood – it takes time and there is a delay of systemic activity

• Protein is also exposed to the local environment containing proteases

• Bioavailability of protein drugs upon s.c. and i.m. administration can be far from 100 % => some diabetics become insulin resistant because of high tissue peptidase activity

• There is not always a direct relationship between plasma level and pharmacological response = not direct pharmacokinetic-pharmacodynamic relationship

• Mechanism of the drug might be complex involving different sequential steps

• Fast clerance of the drug may not necessarily mean that drug action is short lived

• A drug may trigger a reaction – measurable pharmacological effects much later (ex: IL-2 is rapidly cleared from the blood comp. and a pharmacological effect – increase in the blood number of blood lymphocytes is observed long afterwards – days)

• Poor patient compliance • Area of interest: finding alternative to the parenteral

route

B. Current approaches for the oral

delivery of proteins • With the exception of the pulmonary route, all other

option have a low bioavalability • Oral administration: very low bioavalability

because of the enzyme attack in GIT and slow and incomplete penetration through gut wall =

• (exception: oral vaccines containing antigenic protein material – even low uptake levels still deliver sufficient material to lymphoid tissue just below the epithelium – Peyer’s patches to induce stron local and systemic immune response)

• Enteric coating –

• Aim: protection of the protein from the digestion in the stomac

• Problems:

▫ Wide range of pH values and enzymes in GIT

▫ Uncontrolled polymerization of polymers used for enteric coatings during storage

Ways to improve BA of the oral

delivery of proteins 1. Co-administration of protease inhibitors and absorption

enhancers to inhibit enzymatic degradation and improve permeability

2. modification of the physicochemical nature of the macromolecule – to improve membrane permeability and proteolytic stability

3. design of particulate drug delivery system: nanoparticles, liposomes – protect the fragile molecules from enzymatic degradation and harsh environment of GIT

4. mucoadhesive polymeric drug delivery systems – to prolong the residence time at the targeted site

5. site-specific delivery to the colon where proteolytic activity is low

1.a) Use of permeation enhancers

• Co-administration of amphiphilic, low molecular mass permeation enhancer improves absorption of a protein

• Mechanisms: 1. disruption and opening of tight junctions to increase

paracellular permeability 2. decrease in mucus viscosity 3. increase in membrane fluidity specific to their class

• Toxicity issues: disruption of tight junctions can result in unwanted permeation of luminal contents and potentially toxic molecules

Intestinal permeation enhancers Type examples mechanism

sufractants Ionic: sodium lauryl sulfate Disrupt intestinal membrane

Nonionic: polysorbitate, Tween 80

Bile salts Sodium glycholate, sodium deoxycholate

Decrease of mucus viscosity and peptidase activity, phospholipid chain disruption, formation of mixed micelles

Fatty acids Sodium caprate, acyl carnites, oleic acid, lauric acid

Formation of mixed micelles, increase of intracellular calcium levels – contraction of jucnction-associated microfilaments

Chitosan and derivates

N-tromethyl chitosan chloride

Bioadhesion and re´duction of tight junctions integrity

Chelating agents

EDTA, salicylates, citric acid Interfere with the Ca ions present between intestinal epitheloal cells – open tight junctions

Other enhancers

Zonula occludens toxin, PCP-cysteine

Interaction with specific receptors on the surface of epithelial cells = opening og tight junctions

1.b) Protease/enzyme inhibitors

• Bind reversibly/irreversibly the target enzyme • Inactivate it, decrease its activity • Examples:

▫ Aprotinin (inhibitor of trypsin, chymotrypsin) ▫ Soybean trypsin inhibitor (inhibitor of pancreatic

endopeptidases ▫ FK448 (chymotrypsin inhibitor) ▫ Chicken ovomucoid (trypsin inhibitor)

• Indirect enzyme inhibition: ▫ pH modifiers – reduction of optimal environment for

enzyme activity

• Combination of protease inhibitors with permeation enhancers – improve BA

• Safety of inhibotors: it may lead to non-specific interactions with dietary proteins, intestinal mucosal damage and feedback-regulated protease secretion following chronic therapy

2. Modifications of physico-chemical

properties of proteins • Physico-chemical modification of proteins:

▫ By addition of functional groups ▫ By conjugation to polymers (proteins are water

soluble, non-immunogenic, biocompatible, devoid of any biological activity, ideally enhance the intrinsic properties of the macromolecule and not increase its toxicity or minimize its biological activity) Ex: N-(2-hydroxypropyl) methylacrylamide poly(ethylene

glycol)

• To improve proteolytic stability and/or membrane permeability

• To reduce the ability of proteins and peptides to induce an immune response

• Approaches: • PEGylation (covalen attachment of poly(ethylene glycol) to

protein) widely use in parenteral formulations to enhance their therapeutical potential, also in oral delivery ▫ Formation of steric shield that protects macromolecule from

recognition by the body´s immune system, and increase their enzymatic stability, reduction in renal clearance of the protein example: PEGylation of recombinant human granulocyte colony

factor

• hydrophobization: including of more hydrophobic AA or by covalent conjugation with lipophilic moieties – enhance transcellular uptake

example: covalent conjugation of fatty acid – lauric, palmitic and butyric with insulin and desmopressin

• amino acid alterations: replacement of 1 or more L-AA with D-AA

example: analogs of endogenous opioid pentapeptide methionine (Met-enkephalin)

• All these methods - risk of weakening the BA of the protein as a result of chemical modification

3. The use of specialized particulate

formulation vehicles • A) Emulsions: protects proteins from chemical and enzymatic

degradation due to solid-oil-in-water (S/O/W) emulsions which enhance GIT penetration and absorption

• W/O/W emulsion effectively entrap hydrophilic drugd ▫ Ex: Anhydrous reversed micelle ARMs of insulin and phosphatidylcholin

• Self-nanoemulsifying drug delivery systems (SNEDDS): oil, surfactant, co-solvents/co-surfactants, drugs administered in gelatin capsules spontaneously form O/W nanoemulsion upon aqueous dilution by the agitation of the GI fluids, bypass FPE via lymphatic transport, better BA ▫ Ex: thiolated chitosan based SNEDDS for oral delivery of insulin

• Self-double-emulsifying drug delivery system (SDEDDS) ▫ Ex: Pidotimod encapsulation in W/O/W

• Challenges: reduced loading capacity, low physicochemical stability during long term storage

• B) Liposomes: improve membrane permeability of proteins by encapsulating proteins inside aqueous core

• But chemical and enzymatic instability in GIT –> surface coating

▫ Ex: liposomes coated with wheat germ agglutinin (WGA) lectin (binds on the intestinal mucosal membrane) for encapsulation of calcitonin

• Archeosomes: archeobacterial membrane lipids containig diether and/or tetraether lipids – increase stability and enhance immune response – also potential vaccine carrier and adjuvant for effective oral immunization

• 3. Nanoparticles: improve psysico-chemical stability of proteins in GIT by imprisoning proteins in the polymeric matric with a size range of 10-1000 nm (<100 nm can be absorbed across intestinal mucosa)

• Nanoparticles should be non-toxic and non-immunogenic

• Their surface can be decorated with specific ligands for targeting the receptor-mediated transport pathways

• silica nanoparticle coated liposomes – increase protein encapsulation and provides protection against enzymatic degradation

• Challenges: low protein incorporation efficiency, uncontrolled molecule release, susceptibility to particle aggregation, possible accumulation of non-biodegradable polymers in tissues

• Used polymers: • PLGA – poly(lactic-coglycolic acid):

▫ Insulin –loadedPLGA nanoparticles ▫ PLGA nanoparticles coated with chitosan ▫ PLGA particles coated with targeting ligands specific for Mcalls – Con A

lecitins = Con A-coatedPLGA nanoparticles for insulin ▫ Exenatide-loadedPEG-PGLA nanoparticles modified with Fc

• PMMA - poly(methyl methacrylate), • PLA - poly (lactic acid):

▫ Insulin-loadedbloc-copolymer containing PLA and Pluronic P85 (PLA-P85-PLA)

▫ Insulin-loadedPLA-PEG nanoparticles with modifiend Fc • Poly (alkyl cyanoacrylate) • alginate: in combination with chitosan or dextran sulfate :

▫ Chitosan-alginate nanoparticels for insulin • cyclodextrins, gelatin • Chitosan: + opens the tight junctions

▫ Chitosan/insulin nanonparticles tested on mices • and its derivates:

▫ TMC (N-trimethyl chitosan chloride): insulin-loadedTMC ▫ CMC Carboxymethyl chitosan, diethyl methyl chitosan, triethyl chitosan,

lauryl succinyl chitosan

• 4) Microspheres: (1 – 1000 µm): advantage: provision of stability for labile compounds that are easily degraded in the intestinal epithelium

• Mechanisms of release of proteins from microparticles: surface or bulk erosion, diffusion through pores and pulse delivery (ex: luteinizing hormone releasing hormone from PLGA microspheres)

• Limited examples because of incompatibilities

4. Stimuli-responsive hydrogels and

mucoadhesive polymeric systems for

enhanced oral absorption of proteins • A) stimuli-responsive hydrogels: respond to changes

▫ in pH – remains unswollen at gastric pH, release their incorporated protein only in small intestin

▫ ionic streght, ▫ temperature

• Hydrogels for protein delivery: copolymerization utilized to obtain relatively strong but elastic hydrogels ▫ Ex: 2-hydroxyethyl methacrylate, ethylene glycol

dimethylacrylate, N-isopropyl acrylamide, methacrylic acid MAA, poly(ethylene glycol) PEG and poly (vinyl alcohol) PVA

• Examples:

▫ P(MMA-g-EG) and poly[methacrylic acid-grafted-(N-vinylpyrrolidine)] [P(MMA-co-NVP) ] hydrogel system for TNF-α monoclonal antibodies mAb – protects TNF-α from acidic environment of the stomac and release mAb in the small intestine – promising carrier for ptotein drug

▫ Alginate-based hydrogel loeaded with BSA

▫ Xanthan gum/poly (N-vinyl imidazole) hydrogel system loaded with BSA

• B) mucoadhesive polymeric systems – retain their contact with the mucus membrane, enabling prolonged residence time at the absorption site – enteric-coated capsule for oral delivery of insulin

▫ Ex: thiolated polymers

Smart polymers for controlled oral delivery of proteins and peptides

Description

Temperature sensitive polymer Sol-to-gel transition responding to changes in pH from ambient to psysiological temp.: possess low critical solution temp. LCST, or high critical solution temp. HCST

pH sensitive polymer Contains acidic or basic groups that either accept or donate protons in response to environmental pH

Biochemical sensitive polymers Highly sensitive to signals generated by a disease and releases drug in accordance with the magnitude of the signal

5. Site-specific delivery of proteins to

the colon • Colonic delivery appears advantageous for the :

▫ protein absorption because of the prolonged transit time

▫ decreased enzyme activity

▫ higher receptiveness towards permeation enhancers

▫ Alkaline pH – reduced degradation compared with gastric environment

• Challenges: bacterial biofilm barriers

6. Alternative approaches for

enhancing the absoprtion of proteins • Use of eutectic mixtures • Cell penetrating peptides CPPs – protein is

conjugated to a specific protein or peptide (ex: oligoarginine)

• Drug delivery with microneedles-based technology: capsules filled with shielded micro-needles with insulin pass through the hostile environment of the stomach, in inetstinal pH shield coating dissolves and the bare microneedles pierce the intestinal epithelium

Advanced oral protein technology

platforms for clinical preparations • Oramed PODTM (protein delivery) technology for

oral delivery of insulin • Enteric coated oral capsule comprising a protease

inhibitor, absorption enhancer and emulsifier • Transient permeability enhancer (TPETM) system for

oral delivery of octreotide - Octreolin®

• TPETM system: Oily suspension of medium-chain fatty acid salt, embodied by sodium caprylate and matrix-forming polymer in a hydrophobic glyceryl triglyceride medium

• Function by a paracellular mechanism through transcient opening of tight junctions

Eligen ® technology for oral delivery of vitamin B12, calcitoni, heparin and insulin

• Enhance trancellular permeation across intestinal cell membranes

• Based on sodium N-[8-(2-hydroxybenzoyl)aminocaprylate] (SNAC) interact with the drug to form complexes that enable trancellular absorption withiut altering the tight junctions

• ChronotropicTM: a „two-pulse“ colonic device for oral delivery of insulin

• Tablet core surrounded by an inner swellable/erodible hydroxylpropyl methylcellulose (HPMC) layer that is enclosed by an intermediate adjuvant layer and and additional HPMC outer coating

• + protease inhibitor (camostat mesilate) • + absorption enhancer (sodium glycocholate) =

acceptable lag phases between the release of the adjuvant and the protein

• Adjuvant is released prior to the protein, creating a more stable environemnt in advance

• CODESTM (Colon Specific Delivery System) technology for oral delivery of insulin

• Consist of lactulose-containig tablet coated with two acrylic films that displays pH-dependent solubility – inner acid-soluble polymer (Euderagit ® E) and an outer enteric soluble polymer (Eudragit ® L) separated by an HPMC layer

• Enteric soluble polymer protects the tablet from the harsh gastric conditions within the stomach but rapidly dissolves within the intestine

• Acid soluble polymer subsequently protects the tablet through its passage within the alkaline env. of the small intestine

• On arrival within the colon, the bacteria enzymatically degrade the lactulose into organic acid – the pH surrounging tablet is lowered sufficiently to enable the dissolution of the acid polymer and initiate drug release

• GIPETTM (Gastrointestinal Permeation Enhancement Technology) for oral delivery of acyline, heparin, insulin and GLP-1 agonists

• Utilizes medium-chain fatty acids, salts and their derivates to enhance protein absorption from small intestine

• Enteric-coated formulations • AxcessTM oral delivery system for insulin • Oral capsule contains protein and permeation

enhancers and solubilizers to enhance trancellular absorption

• CapsulinTMIR

• PeptelligenceTM technology for oral delivery of salmon calcitonin

• Enteric-coated tablet with permeation enhancer (loosens tight junctions and promotes paracellular trnasport) and citric acid as a main excipient (decreases intestinal pH to prevent degradation of the peptide by intestinal enzymes)

• Multi Matrix MMY Technology for oral delivery of low molecular weigh heparin

• Enteric-coated tablets with pH-resistant acrylic copolymers which delay the release until the tablet reaches the target site

• Controlled release over the lenght of the colon

C. Current approaches for the nasal

delivery of proteins Relative advantage: • Blood-brain barrier (BBB) passage into the CNS –

treatment of many neuronal degenerative disorders • Non-invasive • Readily and easily accesible • Highly vascularized • Avoidance of hepatic FPE • Fast uptake • Proven track record with a number of conventional

drugs • Spatial containment of absorption enhancers is possible • Relatively low activity of proteolytic enzymes • Patient friendly

Relative disadvantage:

• Reproducibility (in particular under pathological conditions)

• Safety (e.g. ciliary movement)

• Low bioavailability of proteins

• 1. enhancers: bile salts, surfactants, fluidic acid derivates, phasphatidylcholines, fatty acids, cyclodextrins, cationized polymer, chelators, cell penetration peptides

• 2. mucoadesive systems: to prolong nasal retention time: carbopol 941, CMC

• + they acts as permeation enhancers opeing tight junctions

1. Polymer-based formulations

a) Nano-/micro-particles:

▫ Ex: Chitosan nanoparticles for the nasal delivery of insulin: chitosa-N-acetyl-L-cysteine and PEG-g-chitosa nanoparticles enhance BA of insulin

b) Nanogels:

▫ Ex: insulin covalently attached to nanogels – poly(N-vinyl pyrrolidine)-based

• Vaccine delivery: cationic cholesteryl-group-bearing pullulan (cCHP) nanogel for tetanus toxoid

• 2. Lipid-based formulation:

• It allows direct nose-to-brain drug delivery – for CNS disorders

▫ Ex: liposomal vaccines ErbB2 T-cytotoxic epitope, the influenza-derives HA T-helper epitope and lipoprotein adjuvant Pam2CAG antilung tumor activity

• Spray-dried polymer-coated liposomes: TMC coated liposomes

D. Current approaches for the

transdermal delivery of proteins Relative advantage: • Easily accessible • Avoidance of hepatic FPE • Removal of formulation is possible if necessary • Spatial containment of absorption enhancers is possible • Proven track record with „conventional“ drugs • Sustain/controlled release possible • Reduced dosing frequency • Sustained drug release • Cost-effectiveness • High patient compliance • Prevent destabilization of protein in the harsh GI

environment • Also for topical drug delivery

Relative disadvantage:

• Low absorption by impermeable stratum corneum

• Barriers: stratum corneum and tight junctions of adhesive membrane proteins in epidermis

• Low bioavalability of proteins

• Enhancers: terpens, glycols (but local irritation and permanent skin damage)

• Nanotechnologies: 1. Polymer-Based Formulations: • Nanoparticles have lipid-fluidizing functions which alter

the extracellular lipids of the stratum corneum ▫ Ex: insulin-loaded-chitosan – transdermal patches ▫ Ovalbumin (OVA)-loaded PLGA – intradermal injections by

hollow microneedles for CD8 and CD4 ▫ Hyaluronan-based disolving microneedles loaded with

PLGA nanoparticles co-encapsulating OVA and poly (I:C) for intradermal immunization

▫ Superoxide dismutase (SOD)-loaded N-trimethyl chitosan nanoparticles in the presence of electret

• Cardiac patch – electrospinning cellulose nanofibers modified with chitosa/silk fibroin multilayers to improve the retention of the engrafted adipose tissue-derived mesenchymal stem cells

• 2. Lipid-Based Formulations

• Emulsions: nanocarriers dispersed in lipophilic vehicles should penetrate stratum corneum – hydrophobic

• Nanoemulsions preparations: high pressure homogenization, phase-inversion temperature, micro-fluidization

• Drawback: creaming and flocculation during long-term storage

• Transcutaneous immunization: safe, non-invasive, economic, patient-friendly, avoids hepatic FPE

▫ Ex: nanoemulsion with FIT-BSA, squalane, Tween 80, Span 80 by high pressure homogenization

▫ Imiquimod solid nanoemulsion

• Insulin-loaded microemulsions: oleic acid, aqueous phase, surfactant phase, dimethyl sulfoxide as a permeation enhancer

• W/O nanoemulsions = precursors to S/O nanodispersions (removal of water and cyclohexane via lyophilization followed by redispersion of the surfactant-drug complex in another oil vehicle)

▫ Ex: transcutaneous immunization vaccine base on S/O nanodispersion: nano-sizesurfactant-OVA complexes dispersed in oil phase

• S/O nanodispersions of BSA

• S/O nanodispersion + arginine-rich peptides as enhancer for insulin

• Liposomes: • Liposomes are easily absorbed by the epidermis and

enhance skin hydration (mixing of the liposome bilayer with intracellular lipids in stratum corneum)

• Liposomal vehicles: Transfersomes® - deformable liposomes or elastic liposomes – gel + insulin or IL-2 and INF-α

• Niosomes: self-assembling elastic nanovesicles • Ethosomes: phospholipids, water, higly concentrated

alcohol: testosterone propionate ethosomes and liposomes

• Invasomes • Incorporation of liposomal formulation into a dissolving

microneedle array ▫ Ex: OVA-PD-Lipos-MNs) ovalbumin/platicodin loaded

liposomes incorporated in microneedle array ▫ Polyvinylparrolidone microneedle array loaded with

antigenand adjuvants encapsulated in liposomes

• 3. Others a) Microneedles: tiny needles with lenghts 20 – 900 µm.

They can pass through stratum corneum to create microchannels

• Painless • Silicon, metals, biodegradable polymers, carbohydrates • First generation: solid micorneedles, now dip-coated

microneedles: • Delivery of recombinant human erythropoetin alfa,

desmopressin, insulin, antigens • Hollow microneedles: fluid drug formulation, pierce the

skin, transport drug through openings • Able to facilitate force-driven fluid flow

• To improve the efficacy of microneedles: use of • Dissolvable polymers – dissolvable microneedles are

completely dissolved upon isertion into the skin – this triggers the release of the drug ▫ Ex: monoclonal IfG-loaded hyaluronic acid-based

dissolving needles ▫ Insulin-loaded microneedle system using poly-γ-glutamic

acid microneedles and PVA/PVP for supporting structures for whole insertion

• Degradable polymers: swell and dissociate the chain network – drug release ▫ Ex: bullet-shapedouble-layered microneedle – water

swellable tips made of polystyrene-block-polyacrylic acid) and insulin

• Incorporation of bio-responsive material: protein release in response to physiological signals (variations in pH, Glc, reactive oxygen species and enzymes) ▫ Ex: bio-inspiredglucose-responsive insulin delivery system

b) Iontophoresis • Uses mild electric current to the skin – charged drug

moves away from the electrode with the same charge into the skin by electro-repulsion

• depends on: size limit of the drug (10 – 15 kDa), overall charge, structure, lipophilicity ▫ Ex: INF alpha2b iontophoration at rats

c) Electroporation • Applying of high voltage electronic pulses for short

duration of time to encrease skin permeability by opening aqueous pores in a revesible manner ▫ E: insulin, parathyroid hormone

E. Current approaches for the

pulmonary delivery of proteins Relative advantage: • Lungs – rapid and high drug absorption due to large surface,

very thin alveolar epithelium thickness and large blood supply • Avoidance of hepatic FPE • Non-invasive • Efficacious at lower doses • Local or systemic delivery • Low enzymatic activity of lung tissue, many immunological

properties • Relatively easy to access • Proven track record with „conventional“ drugs • Substantial fractions of insulin are absorbed • Lower proteolytic activity that in GIT

Relative disadvantage: • Reproducibility (in particular under pathological

conditions – smokers/non-smokers) • Safety (e.g. immunogenicity) • Presence of macrophages in the lung with high

affinity for particulates • Short duration of activity due to the rapid removal

of the drug, phagocytosis by alveolar macrophages

• Key factor: delivery device: nebulizers, metered-dose inhalers, dry powder inhaler

1. Polymer-Based Formulations • Widely used • Chitosan, alginate, gelatin, poloxamer, PLGA,

polyehtylene glycol PEG ▫ Ex: BSA loaded Nps using poly(glycerol adipate-co-ω-

pentadecalactone) ▫ PLGA and gelatin NPs for pulmonary protein/DNA

delivery • 2. Lipid-Based Formulation • The most effective pulmonary carrier for proteins • Solid lipid nanoparticles (SLN)

▫ Ex: papain in mannitol and trehalose SLNs

F. Current approaches for the occular

delivery of proteins Relative advantage: • High potency • Less toxicity • Low drug-drug interactions • Increased biological and chemical diversity Relative disadvantages: • Degradation • Low permeabilty • Short in vivo half-life • Risk of immunogenic effect

• Chemical chaperones (protein aggregation inhibitors) • Co-administration of recombinant hyaluronidases

(increse the penetration) 1. Polymer-Based Formulations a) Nanoparticles: topical, periocular, suprachoroidal,

intravitreal routes • Problems: loss of bioactivity, low stability, extensive

nanoformulations • Only few systems:

▫ Ex: polymer-based thermo-gelling system: tehrmosensitive hexanoxyl glycol chitosan carrier for brimonidine

▫ Pentablock copolymer (PB-1: PCL-PLA-PEG_PLA-PCL)-based nanoformulation suspended in a thermosensitive gelling copolymer (PB-2: mPEG-PCL-PLA-PCL-PEGm) for IgG-Fab

• b) Polymeric Micelles: amphiphilic block copolymers with hydrophilic chains forming a shell and hydrophopbic chains forming a core

• poloxamer 407, 188 methoxy poly(ethylene glycol)-poly (ε-caprolactone), poly (butylene oxide)-poly (ethylene oxide)- poly (butylene oxide), polyhydroxyethylaspartamide, N-isopropylacrylamide

• Improved stability, high drug loading capacity, low risk for toxicity, high modifiable surfaces, controlled drug release, high patient compliance ▫ Ex: anti.Flt1 peptide conjugated to tetra-n-butyl

ammonium modified hyaluronate • New technologies: micelles as nano-scale

microbubble precursors, stimuli-responsive micelle (t, pH, light, magnetic fields, disease specific biological components)

c) Dendrimers: nano-sized polymeric carriers that entrap and/or conjugate high molecular weight molecules

• Radially symetric molecules with well-defined, homogenous, and monodisperse structure consisting of tree-like branches

• Based on polyamidoamines (PAMAM) most common, commercially available, polyamines, polyamides, poly (aryl ethers), polyesters, carbohydrates

• Cationic dendrimers – max. absorbtion (interaction with lipid bilayer)

• Multiple functional groups on their surface – possible to target any location in the body ▫ Ex: hybrid PAMAM dendrimer hydrogel/PLGA

d) Others: • Nanowafers – tiny transparent circular or rectangulate

membranes that contain arrays of drug-loaded nanoreservoirs that release drug in a highly controlled manner and for a longer duration than eye drops

• Composed of various polymer: PVA, PVP, HPMC and CMC

• Applied with patient fingertip, withstand blinking • Slow drug release, slowly dissolves

▫ Ex: PVA-based nanowafers loaded with axitinib

• Drug loaded contact lenses: slow diffusion of drug, longer drug rsidence time on the eye

2. Lipid-Based Formulations • Risk of ocular complications as retinal detachment, cataracts

▫ Ex: pigment epithelium-derived factor loaded immuno-nanoliposomes (PEDF loaded INLs)

• Solid-lipid nanoparticles (SLNs): stability, targetability, controlled release, ease scale-up, non-toxicity, improve BA of hydrophilic and lipophilic drugs, can be sterilized by autoclave, dispersible in aqueous media

• But low drug loading efficiency, initial burst release of drug, rapid elimination ▫ Ex: SLN system for p-IL10

• Niosomes: self-assembling nanovesicles composed of non-ionic surfactants that behave like liposomes

• Stable, biodegradability, biocompatibility, lack of immunogenicity, low toxicity

• Discomes – niosomes woth large structure created by the addition of Soluan C24 (non-ionic surfactant)

• Early stage of development

G. Current approaches for the rectal

delivery of proteins Relative advantage: • Relatively easy accessible • Partial avoidance of hepatic FPE • Probably lower proteolytic activity that in the upper

parts of GIT • Spatial containment of absorption enhancers is possible • Proven track record with a number of „conventional“

drugs • Improve BA of protein drugs which are very vulnerable

to degradation Relative disadvantage: • Low bioavalability of proteins

• Enhancers: for insulin, heparin, calcitonin, rhGCSF, human chorionic gonadotropin

• Can cause membrane damage and irritation

• Protease inhibitors

• Prodrugs: protect proteins against degradation from peptidases and othe enzymes

• 1. Polymer-Based Formulations a) Hydrogels:

▫ Ex: hydroxypropal methyl cellulose-co-polyacrylamide-co-methacrylic acid hydrogel (HPMC-co-PAM-PMAA) as a rectal suppository loaded with insulin

b) Nanoparticles: chitosan, PLGA, PLA, methacrylic acid + surface modification Ex: complex of galactosylated low molecular weight chitosan

and antisense oligonucleotide with THFα

Surface modification of PLGA nanoparticles with PEG in non-covalent fashion

Carbo nanotubes CNTs: cyllindrical shape, easy surface modification, superior mechanical properties, high termal conductivity, ability to penetrate cell membranes Ex: multiwalled carbon nanotube complexes loaded with 2-

arachidonoylglycerol – new pathways

• 2. Lipid-Based Formulations

• Ex: nanoliposomes containing hepatitis B surface antigen

• Solid lipid nanoparticles – but without proved advantage over conventional formulations

H. Current approaches for the buccal

delivery of proteins Relative advantage: • Easily accessible • Avoidance of hepatic FPE • Probably lower proteolytic activity that in the lower

parts of GIT • Spatial containment of absorption enhancers is

possible • Option to remove formulation if necessary Relative disadvantage: • Low bioavalability of proteins, no proven track

records yet (?)

Release control

• If ther is a direct relationship between blood level and therapeutic affect – it is important to maintain therapeutically relevant drug concentrations in the bood stream (many proteins are short-lived in the blood)

• = portable pump systems with adjustable pump rates are available (catheters provide a link between the pump and for. ex. peritoneal cavity

• Particularly useful if a constant dose input is required and the drug is needed over a limited period of time

• For insulin (plasma half-life 5 min) s.c. injections: • amorphous insulin plus Zn2+ ions = moderate

prolongation of drug action • Crystalline insuline + Zn2+ ions = long-acting

product • Addition of protamine to insulin and Zn2+ ions =

protracts the insulin effects even more (72 hous) • Isophane insulin (NPH: neutral protamine

Hagedorn) contains insulin and protamine in isophane proportions (no excess of either component) – intermediate-acting formulations

• At present, efforts are being made to build „closed-loop“ systems in which administration of insulin is controlled by:

• A biosensor permanently monitoring blood Glc • An infusion pump with adjustable pump rate • An electronic section with algorithm linking

blood Glc levels to insulin need at any time • Available in hospital to stabilize blood Glc for

limited periods, no portable systems for chronic use

Development

• Artificial pancreas: isolated insulin-producing β-cells from the islets of Langerhans are introduced into the body in a container system with a wall that allows the passage of Glc, insulin and nutritients however the wall keeps the β cells separated from the patient’ immune system

• Increasing blood Glc levels -> stimulation of the secretion of insulin -> insulin is released from the container -> normal blood level of Glc