protein drug delivery and gene drug delivery

protien drug delivery 1

PROTEIN DRUG DELIVERY

PRESENTED BYSUBODH S SATHEESH

MPHARMPHARMACEUTICS

ECPS


Rise as a potential drug Treatment of many diseases Recombinant technology protein market

INTRODUCTION


Primary Secondary Tertiary quaternary

Structure of protein


Classification by functions of proteins: Enzymes : DNA and RNA polymerase Hormones : Endorphine and encephalin Transport proteins : Cytochrome C, Albumin, Haemoglobin. Antibodies : Interferon, Fibrin. Structural proteins : Collagen, Elastin. Motor proteins : Actin, Myosin. Signalling proteins : GTPase. Storage proteins Egg albumin, milk casein. Classification of proteins by location in the living cell: Membrane proteins

Internal proteins External proteins Virus proteins Classification of proteins by post translational modification:

Native protein Glyco protein

Classification of proteins


Solubility Partition coefficient Self aggregation Hydrogen bonding Association

Physico chemical properties of peptides


1.Denaturation 2. Adsorption 3. Aggregation and Precipitation . Chemical Instability: 1. Deamidation . 2.Oxidation and Reduction . 3. Proteolysis 4.Disulfide exchange 5. Racemisation

Instabilities of proteins


Enzymatic barrier Limits absorption of protein drugs from G.I. tract.

Intestinal epithelial barrier Involved in the transport of protein drugs across the intestinal epithelium

Capillary endothelial barrier Involved in transport of protein drugs across the capillary endothelium.

Blood brain barrier

Barriers to protein drug delivery


Parenteral systemic delivery: Non‐parenteral systemic delivery: a. Oral route b. Nasal route c. Buccal route d. Ocular routee. e. Rectal route f. Transdermal route g. Pulmonary route

Routes of administration


Most effective method of delivery intravenous(IV), intramuscular(IM), subcutaneous(SC Biomedical applications

Parenteral protein drug delivery


Insulin Lente semilente ultra lente Needles and infusion pumps Vasopressin Covering a section of microporous

polypropylene(Accurel) tubing with collodion a long lasting and constant in‐vitro release

Biomedical preaparations


a. Oral route b. Nasal route c. Buccal route d. Ocular routee. e. Rectal route f. Transdermal route g. Pulmonary route

Non parenteral route


Patient complaince Strategies Promote adsorption

Oral route


Bonding of (PEG) and alkyl groups fatty acid radicals to produce desired amphiphilic oligomers oligomers are conjugated to proteins or peptides to obtain desi

red amphiphilic products can resist excessive degradation of protein or peptide drugs technology reduces self‐association, increases penetration

and increases compatibility with formulation ingredients than parent drug

Nobex technology


protection against the metabolic barrier in GIT absence of a carrier system for

absorption of peptides with more than three amino acids

Proteins are labile due to susceptibility of the peptide backbone to proteolytic cleavage

Prodrug approach olefenic substitution, d‐amino acid substitution,

dehydro amino acid substitution, carboxyl reduction

Problems associated with oral delivery


Pulmonary protein delivery offers both local targeting for the treatment of respiratory diseases and increasingly appears to be a viable option for the delivery of proteins systemically

The lung is easy to access, has decreased proteolytic activity compared with the gut

Careful choice of carrier and device can facilitate delivery to a specific area of the lungs.

The only protein for inhalation currently available on the market is Dnase

Inhaled insulin leuprolide and gamma‐interferon are in trials.

Nasal delivery


Insulin: The nasal absorption of insulin is increased by coadministration of bile salts

inhibition of respiratory virus infection was studied by intranasal administration of human leukocyte interferon.

Nasaldeliveryof oligopeptides: Examples: Dipeptides: 1‐tyrosyl‐1‐tyrosine and its methyl

esters Tripeptides: Thyrotropin‐releasing hormone(TRH) Pentapeptides: Leucin‐enkephalin, met‐enkephamide

Nasal delivery of protiens


Advantages of nasal route: Convenient,simple,practicalway of drug administration The high vascularization permits better absorption. First pass metabolism can be avoided. Rapid onset of action. Disadvantages of nasal route:

Long term use may lead to toxicity to mucosa. During disease states (e.g. common cold) some alteration in the nasal environment may occur

Advantages and disadvantages


it offers excellent accessibility and avoids degradation of proteins and peptides.

Various types of polymers like sodium CMC, hydroxypropylmethyl cellulose, PVP, acacia,calcium carbophil are used for delivery of proteins or peptides via buccal route.

1)Adhesive tablets: e.g. Adhesive tablet based on hydroxypropylcellulose.

2)Adhesive gels: e.g. By using polyacrylic acid and polymethacrylate as gel forming polymers.

3)Adhesive patches: e.g. Protirelin in HEC patches and buserelin

Buccal route


It is robust, much less sensitive to irreversible irritation even on long term treatment.

Absence of enzymatic barrier. Well acceptable to the patients. Easy accessibility administration as dosage forms. It is

attached or removed without any pain or discomfort

Advantages


The ocular route holds immense potential for peptides or proteins intended for pathological ophthalmologic conditions

The ocularroute is the site of choice for the localized delivery of opthalmologically active peptides and proteins for the treatment of ocular disease that affect the anterior segment

tissues of eye. The use of nanoparticles, liposomes, gels, ocular inserts, bioad

hesives or surfactants are necessary to enhance ocular absorption of proteins or peptides

Occular route


Affect aqueous humor dynamics: Calcitonin gene related factors, LHRH, vasopressin

Immunomodulating activities: Cyclosporine, interferons.

Act on inflammation : Substance P, enkephalins. Affect wound healing:Epidermal growth factor, fibro

nectin

Proteins or peptides with opthalmological activities


It is one of the recent ideas The coadministration of an absorption promoting

adjuvants such as sodium glycocholate, has been reported to enhance the rectal absorption of insulin.

Bile salts, such as sodium salts of cholic, deoxycholic and glycocholic acids, have also been shown to enhance the rectal absorption of insulin

Vasopressin and its analogs,pentagastrin and gastrin, calcitonin analogs and human albumin have been investigated for rectal delivery of protein or peptide based pharmaceuticals

Rectal route


It is highly vascularized. It avoids first pass or presystemic metabolism. Drug can be targeted to the lymphic system. It suitable for drugs that cause

nausea/vomiting and irritate GI mucosa on oral administration.

A large dose of drugs can be administered.

Advantages of rectal delivery


Transdermal delivery has attracted considerable interest as a route for administering peptides and proteins.

The small peptides such as thyrotropin releasing hormone (TRH) vasopressin, have great difficulty in permeating the skin barrier.

Percutaneous absorption of elastin peptides has shown better distribution.

Transdermal route


Iontophoresis It is use of electric current to drive charged drug molecules into skin by placing them under an electrodeof like charged

DC iontophoretic device, as the power source for direct current and were able to deliver insulin transdermally to diabetic hairless rats, with attainment of a reduction in hyperglycemia

Pulse DC iontophoresis: By delivering a pulse current with a 20% duty cycle (4µsec), followed by an 80% depolarizing period(16µsec), a β‐blockers was successfully delivered systemically human subjects without polarization induce skin irritation.

Approaches in transdermal route


In this method ultrasound is applied via a cupling contact agent to the skin.

Insulin, IFN γ, erythropoietin can be delivered by this method Surfactants and azone have been used for topical delivery of p

eptide or proteins Prodrugs; Prodrug with modeled physicochemical

characteristic permeated well across the skin. LHRH, TRH, neurotensin can be delivered by this method.

Penetrationenhancers: Penetration enhancers like oleic acid, dimethylsulphoxide are used.

phonophoresis


Advantages of transdermal route: Avoids the hepatic first‐pass effect and gastrointestinal breakdown. Provides controlled and sustained administration particularly

suitable for the treatment of chronic disease. Reduces side‐

effects, often related to the peak concentrations of the circulating agent; Enables self‐administration and improves patient compliance, due to

its convenience and ease of use. Permits abrupt termination of drug effect by simply removing the

delivery system from the skinsurface. Limitations of transdermal route: A low rate of permeation for most of protein drugs due to their large

molecular weight. High intra‐ and inter‐

patient variability. Because the skin has a relatively low proteolytic activity, the peptide drugs have poor skin permeability.

Advantages and limitations


GENE DRUG DELIVERY


successful design of a gene delivery system requires complete understanding of the interaction mechanism between the target cell and delivery system

Cell targeting refers to delivery of the therapeutic agent to a specific compartment or organelle of the cell

Endocytosis gene therapy cellular release takes place to initiate DNA

transcription and translation, and to produce the related protein

Introduction


Viral drug delivery Non viral drug delivery Physical methods Chemical methods

Gene drug delivery


It consist of viruses that are modified to be replication-deficient which were made unable to replicate by redesigning which can deliver the genes to the cells to provide expression

Viral systems have advantages such as constant expression and expression of therapeutic genes

Limitations are use of viruses in production, immunogenicity, toxicity and lack of optimization in large-scale production.

current gene technologies concentrates on the use of viral vectors that provide high transduction effectiveness and advanced level of gene expression.

Viral gene delivery systems


Adenoviral systems Retroviral systems Lentiviral systems

Viral GDS


Adenoviruses (Ad) were first discovered in 1953 by isolation from human adenoid tissue cultures.

Commonly used as gene vectors adenoviruses Ad2 and Ad5 are The most widely studied

adenoviruses. The capsid of an adenovirus determines virus tropism. Adenoviruses are well-characterized, non-integrated, 26–

40 kb in length, relatively large, non-enveloped, linear dsDNA viruses coated with icosahedral particle, with a diameter of 950 Å (excluding elongated fiber proteins) and a molecular weight of approximately 150MDa

Adenoviral systems


in Figure it was reported that crystal structures of single Ad proteins contained fiber knob, shaft, domains, penton base, hexon, and cysteine protease.

Ad capsid consists of 252 sub-units called capsomeres, which contain 240 hexonproteins and 12 of the penton base.

In addition, the capsid contains pIIIa, pVI, PVIII, and pIX proteins. Each of the 12 capsid corners contains penton bases wrapped by 5 hexons.


Instead of combining its own DNA with the genomes of the host cell, the adenovirus, remains as an episome within the infected cell.

Penton and fiber proteins of virus capsid interact with the coxsackievirus-adenovirus receptor cell surface protein to provide cell binding

Viral capsid proteins dissociate prior to endocytosis, and the pH value of the viral endosome decreases due to proton pumps

For successful delivery of DNA to the nucleus, viruses must facilitate cell-specific binding, endocytosis internalization, propagation from endocytic vesicles to cytosol, delivery into cytoplasm

Mechanism


Retroviruses are diploid, single-stranded, circular-enveloped RNA viruses of the family Retroviridae, with a genome of 7–11 kb, and a diameter of approximately 80–120 nm

Retroviruses cause diseases such as AIDS, leukemia, and cancer

Retroviruses are viruses that integrate with host genome to produce viral proteins (gag, pol, env) that are extracted during gene delivery.

Commonly used retroviruses are the Moloney murine leukemia virus species, which have the capacity to deliver exogenous genetic material up to approximately 9 kb.

Retroviral systems


An ideal retroviral vector for gene delivery should be cell-specific, regulated, and safe.

Retroviruses have a lipid envelope. In order to enter a host cell, they use the interactions between cellular receptors and virally encoded proteins, which are embedded in the membrane

CKRs are a family of cell-sfurface-G-proteins functioning as receptors to stain molecules called chemokines

Retroviruses introduce their genetic material to the host cell genome in a stabile manner during mitotic division


Most retroviruses infect cells that can be actively divided during mitotic division. This property protects normal tissue, and although it naturally targets the tumor

A retrovirus infects the target cell by providing interaction between viral envelope protein and cell surface receptor on the target cell

Many types of retrovirus types require degradation of mitosis and then the nuclear envelope for the arrival of a viral genome within the nucleus.


Lentivirals are viral systems without small, retrovirus-like viral proteins and no capacity for replication

The most important advantage of lentiviruses compared with other retroviruses is their ability for gene transfer to non-dividing cells

Genome of lentiviruses have a more complicated structure; they contain accessory genes which regulate viral gene expression, control combination of infectious particles, modulate viral replication in infected cells, and are associated with the continuance of infection.

Lentiviral systems


HIV-1 is one of the most widely used lentiviral vectors, and contains six accessory genes (tat, rev, vif, vpr, nef, vpu). These proteins are involved in all steps of cell cycles, which are termed: budding, maturation, and integration.

Lentiviral vectors do not require degradation of the nuclear membrane for integration.

Lentiviruses that are encoded with the Gag matrix protein integrase enzyme and vpr protein interact with the nuclear import mechanism of the target cell and manage active transport of pre-integration complex via nucleopores.

Receptors have been defined for many retroviruses. The best-characterized example is CD4 molecule, which serves as a receptor for lentiviruses including HIV.


many studies that used viral vectors reported unsatisfactory results, due to the immunologic and oncogenic adverse effects of these vectors.

It is overcome by NVGDS non-viral vectors have many advantages, such as easy of

fabrication, cell/tissue targeting, and low immune response biggest disadvantage of non-viral vectors in clinical use is low

transduction efficiency. the biggest difficulty in gene therapy is the development of

physical methods to ensure gene transfer to target cells of the gene delivery vectors and delivered gene.

Non viral gene delivery systems


Compared to viral delivery systems, non-viral carriers are less toxic and 450 immunogenic non-viral vectors is ease-of-production.

A number of barriers need to be overcome in order to increase the effectiveness of non-viral vectors in humans. These barriers are classified as production/formulation/storage; extracellular barriers; and intracellular barriers

Anatomic barriers are extracellular matrixes coating the cells, which prevent direct transport of macromolecules to target cells through epithelium and endothelial cell sequences


The most critical barrier to effective DNA transfection was regarded as the transition of plasma membrane. Typically, naked nucleic acids cannot cross cell membrane by cellular uptake mechanisms such as endocytosis, pinocytosis, and phagocytosis

Physical approaches, including electroporation, gene gun, ultrasound, and hydrodynamic delivery are based on the application of a force to increase the permeability of the cell membrane and promote intracellular gene transfer


Two of the most important advantages of synthetic carriers are that they do not display immunogenicity, and large-scale production is easy

Non-viral vectors can trigger an inflammatory response, since they do not provide a specific recognition

they are much less dangerous than viral vectors in terms of antigen specific immune response

Non-viral vectors should be designed according to specific cell targeting; cellular uptake and and potential immune response should be minimized.


Gene guns Electroporation Ultrasound polymers

Physical methods


Delivery with gene gun method is also termed ballistic DNA delivery or DNA-coated particle bombardment, and was first used for gene transfer to plants in 1987.

This method is based on the principle of delivery of DNA-coated heavy metal particles by crossing them from target tissue at a certain speed

Generally, gold, tungsten or silver microparticles were used as the gene carrier

Gene-gun-based gene transfer is a widely tested method for intramuscular, intradermal and intratumoral genetic immunization.

Gene gun


It does not use toxic chemicals or complex biological systems

delivery is achieved without the need for a receptor DNA fragments of various sizes, including large ones,

are transported, there is no need to introduce foreign DNA or protein.

it has high repeatability, production of heavy metal particles is easy .

However, in this method, gene expression is short-term and low.

Advantages


Ultrasound has many clinical advantages as a gene delivery system, due its easy and reliable procedure

Microbubbles or ultrasound contrast agents decrease cavitation threshold with ultrasound energy.

Mostly perfluoropropane-loaded albumin microbubbles were used.

The transfection efficiency of this system is based on frequency, time of ultrasound treatment, the plasmid DNA mount used, etc

ULTRASOUND


Electroporation includes controlled electric application to increase cell permeability

Electroporation introduces foreign genes into the cell by electric pulses. In this method, pores are formed on the membrane surface to enable the DNA to enter the cell.

If the molecule is smaller than the pore size , it can be transferred to the cell cytosol through diffusion

loaded molecules and ions can be transported from the membrane via electrophoretic and electro-osmotic means via the effect of electric regions

Electroporation


Polymers are long-chained structures composed of small spliced molecules called monomers.

Polymers that are composed of a repeated monomer are called homopolymers, while those composed of two monomers are called copolymers.

Biodegradable polymers are non-water soluble, and undergo chemical or physical change in biologic environment.

Polyamides, dextran, and chitosan are examples of biodegradable polymers

non-biodegradable polymers are not degraded in biological environments; hydrophilic polymers are hydrogels, which are non-water soluble and

swell in water, while hydrophobic polymers are non-water soluble and do not swell.

polymers


hydrophilic hydrogel polymers include PVA, polyvinylacetate, polyethyleneglycol, polyacrylic acid.

hydrophobic polymers include silicones, and polyethylene vinyl acetate

ethyl cellulose (EC), hydroxypropyl methyl cellulose (HPMC), cellulose acetate phthalate (CAP), and eudragit derivatives are commonly used in controlled release systems

For polymer selection, in addition to its physicochemical characteristics, characterization of extensive biochemical characteristics and preclinical tests are required to demonstrate its reliability.


Encapsulation Electrostatic interaction Liposomes dendrimers

Chemical methods


Most of the polymeric vectors presently in use form complexes with negatively charged DNA by electrostatic interaction

At adequate nitrogen– phosphate ratio, the polymer and the DNA form nanocomplexes, which allows both cellular DNA uptake and also protects the DNA from nuclease enzyme.

Electrostatic interaction


An alternative to electrostatic condensation of DNA is encapsulation of DNA with a biodegradable polymer.

Polymers that have an ester linkage in their structures (like polyesters) are hydrolytically degraded to short oligomeric and monomeric compounds, which are more easily discharged from the body.

The degradation mechanism and DNA release can be controlled by changing the physicochemical characteristics and composition of the polymer.

DNA is protected from enzymatic degradation by encapsulation.

Encapsulation


Remington the science and practice of pharmacy volume 1

Nejm Gene therapy and novel drug delivery page 1-36 Intechopem Gene therapy and viral and nonviral

vectors 387-402 Ijcpr protien drug delivery volume 3 285-327 Rhienberg Protien drug delivery 1-17

REFERENCES

58protien drug delivery

protein drug delivery and gene drug delivery

Science

protien drug delivery

pulmonary protein delivery

pulmonary route

functions of proteins

structural proteins

motor proteins

signalling proteins

buccal route