Overview of types of pharmaceuticals (Proteins, peptides nucleic acid, antibodies based drugs)

Peptides and proteins are a new and upcoming therapeutic in the form of biopharmaceuticals that can be used in a range of different diseases. We classify proteins as having chains of more than 100 amino acids while peptides have less than 50 amino acids both have advantages and disadvantages as therapeutic drugs. These proteins/peptides have been increasing in interest since Lypressin a vasopressin analogue was first made available in 2005.¹ Since then proteins and peptides have grown to constitute almost half of the drugs currently being investigated by pharmaceutical companies.² To date, more than 100 proteins have been approved as therapeutic drugs and much more are in the approval process by the FDA.³ These recombinant proteins are used as antibody-drug conjugates, vaccines and enzymes to name a few and have a wide range of uses from diagnosis to disease management or cure. They have been proved treatments for many diseases such as heart disorders, diabetes and cancer.^3-5 However, there is a range of problems associated with delivering peptides in-vivo due to the induced immune response and biodegradability.¹

Have a focus on proteins and peptides

We can classify therapeutic proteins and peptides as done by Leader et al in figure 1 showing each category in terms of application and function based on physical and biochemical properties.⁵ These groups as stated are group 1 which refers to an enzymatic or regulatory activity, group 2 relating to specific targeting activity, Group 3 refers to vaccines while group 4 is for diagnostic therapeutics. The area we are interested in is group 1 and 2 proteins/peptides for targeted regulatory or enzymatic reactions a common peptide from this group is insulin for diabetes treatment a regulatory disease.⁵

Figure 1 Classification of therapeutic proteins⁵

Therefore, to deliver proteins and peptides to the bloodstream of a mammal in significant and active states, a multipurpose system must be developed. This will enable these therapeutic proteins and peptides to be protected and delivered through the intestinal system.⁶

Table of best selling proteins and peptides per year

The U.S Food and Drug Administration(FDA) has approved 62 therapeutic proteins between January 1, 2011, and August 31, 2016. With 48% of these therapeutics being monoclonal antibodies, of these antibodies coagulation factors were the largest group with 19% of the total approved therapeutic proteins. The second largest group was replacement enzymes at 11% of all proteins approved, the remaining 22% was made up of hormones, fusion proteins plasma proteins and growth factors.⁷ The range of proteins approved by the FDA help to treat a large area of diseases with 26% of the approved therapeutics used for oncology, and 29% used for hematology. With Dermatology, endocrinology, genetic disease, immunology, infection disease, musculoskeletal, ophthalmology, rheumatology, cardiology/vascular disease, gastroenterology and nephrology making up the remaining 45%. This goes to show that these approved therapeutics can treat a wide range of patients and have a huge benefit to the medical industry. The breakdown showing the year by year analysis of how many proteins were approved by the FDA can be seen in Fig. 2 as well as the above percentages shown in a compact format.

Figure 2 U.S. Food and Drug Administration (FDA)-approved therapeutic proteins (2011–2016*). (a) Bar graph showing the number of therapeutic protein FDA approvals by year (2011–2016*). (b) Pie chart showing the distribution of FDA-approved therapeutic proteins (2011–2016*) by drug class. (c) (Left) Pie chart showing the distribution of FDA-approved therapeutic proteins (2011–2016*) by therapeutic area. (Right) Pie chart showing the distribution of secondary therapeutic area for oncology drugs. *January 1, 2011, through August 31, 2016. ⁸

General routes of administration (we are focusing on oral delivery)(Injection, Oral, Transdermal, inhaled, nasal) ????

Therapeutic proteins and peptides like other drugs have a number of routes of administration these include Drugs are introduced into the body by several routes.

• Taken by mouth (orally)
• Given by injection into a vein (intravenously, IV), into a muscle (intramuscularly, IM), into the space around the spinal cord (intrathecally), or beneath the skin (subcutaneously, sc)
• Placed under the tongue (sublingually) or between the gums and cheek (buccally)
• Inserted in the rectum (rectally) or vagina (vaginally)
• Placed in the eye (by the ocular route) or the ear (by the otic route)
• Sprayed into the nose and absorbed through the nasal membranes (nasally)
• Breathed into the lungs, usually through the mouth (by inhalation) or mouth and nose (by nebulization)
• Applied to the skin (cutaneously) for a local (topical) or bodywide (systemic) effect
• Delivered through the skin by a patch (transdermally) for a systemic effect

Proteins with oral delivery

There are 3 key issues with oral delivery of proteins and peptides

• Proteins/peptides are unable to survive the stomach pH and saliva enzymes causing degradation and denaturation of the protein/peptide. ⁹
• The small intestine of mammals has several proteases (E.g Trypsin, Chymotrypsin and pepsin) all of which destroy the structure of proteins and peptides denaturing them.¹⁰
• Once protein cross the small intestinal epithelial cells they undergo intestinal first-parse metabolism in the liver and are destroyed.¹¹

The lack of oral bioavailability means that an injection is required, in addition, the risk of immunological effects in the human body provides a further problem for delivery and efficacy of therapeutic peptides.  This can lead to antibodies being created by the immune system to counteract the therapeutic proteins or peptides being introduced. This follows B- and T-cell epitopes that can further reduce the effectiveness of proteins by attacking and breaking down any proteins introduced, this process becomes more prevalent over time.¹²  A large amount of research has been done to try and provide an alternative delivery system to protect against denaturing processes including enzymatic and acidic hydrolysis.¹² For a specific protein/peptide to achieves an effective delivery outcome, we need to investigate how we create the therapeutic formulation, route of administration as well drug endpoint in the in-vitro system.

Nanoparticles as delivery vehicles

Lipidsomes are formed by cationic lipids with the hydrophobic tail are buried inside the bilayer as seen in figure 2. The curvature of these bilayers similarly to micelles cause them to form spherical particles with a hydrophilic core and otter layer separated by a hydrophobic bilayer.⁷ ????????? should I go into more detail about liposomes and micelles here

Figure 3 Different lipidic nanoparticles showing micelles(A), liposomes (B) and inverse micelle (C).⁷

The general paragraph on a range of NP’s

A range of nanoparticles have been developed to attempt to deliver protein/peptide-based drugs orally these include gold, polymer, lipid and chitosan nanoparticles.^{8, 13-18} They have so far been unable to bypass all 4 main factors that negate protein uptake into the bloodstream after oral ingestion as stated previously.¹⁰ Certain nanoparticles such as chitosan have been shown to survive both the stomach conditions and also have the ability to protect the encapsulated enzyme from digestion in the small intestine.¹⁶ Though these nanoparticles are unable to survive first parse metabolism in the liver after they cross the small intestine epithelial cells. The highest efficiency for oral delivery has come in the form of lipid-based nanomaterials/nanoparticles with liposomes and micelles showing some success with ?? to ??% efficacy compared to subcutaneous injection.^{19, 20}

Details on lipid nanoparticles(Coming up on 6 pages)

A new novel way to encapsulate and transport these peptides in a biological system is to use LCP (Lipid Cubic Phase) in either bulk or dispersed form(cubosomes).²¹
These bilayers and their unit cells can be seen in figure 3 the number of channels can be seen to be 3 in the Q_II^G(Gyroid) 4 in the Q_II^D (Diamond) and 6 in the Q_II^P (cubic bulk structures). This unit cell is still present in the cubosome structure which is the dispersed form of the bulk phase biocontinuous layers. These cubosomes are found to be approximately 100-200nm are act as a very effective therapeutic protein carrier and a novel delivery system.

Figure 4 Cubic phase lipids form different structures depending on the lipid curvature and packing number as seen above QII^G with space group Ia3d(Left), QII^D with space group Pn3M(centre) and QII^P with space group Im3m(right).²²

The adaptability of this system is in the fact that hydrophilic, hydrophobic and amphiphilic therapeutic proteins can be incorporated into the cubosomes. This is due to the hydrophilic water channels being able to capture and hold the hydrophilic proteins with the hydrophobic proteins being embedded in the lipid bilayer.²³ The amphiphilic proteins will be able to be incorporated into both the water channels and the lipid bilayer. These therapeutic peptides and proteins also retain their active state when incorporated into the lipid layer due to the fact that the lipid bilayer mimics the cellular membrane.²² This process occurs due to the fact that proteins and peptides are designed to be stable and active in cellular membranes. Most proteins need to interact with this membrane in nature at some point, especially when they are receptor binding peptides or proteins.

The addition of amphiphilic additives such as detergents, sterols, single and multi-chain amphiphiles have been shown to affect the lipid nanostructure.  In a similar way, the addition of amphiphilic and hydrophobic proteins and peptides can significantly impact the LCP nanostructure and, at higher protein concentrations, can disrupt it completely.^23-25  This has profound implications for the end use of such materials in drug delivery.
For example, Phan et al have demonstrated that release rates depend strongly on the lipid nanostructure.²⁶

Endocytosis and stuff ++

There are 5 main types of endocytosis which have been thoroughly investigated previously it is also found that the cell type plays a large role in endocytosis.²⁷ Phagocytosis is only available to certain cells such as macrophages, monocytes, neutrophils, and dendritic cells with this process it is possible to endocytose large nanoparticles from 1000-2000nm.²⁷
Clathrin-mediated endocytosis(CME) is the second route as is relevant for cellular nutrient uptake and cellular membrane components such as cholesterol. This process uses non-specific pathways for lipids such as cholesterol or receptor-specific pathways for certain proteins and nutrients.²⁷ This process caters to smaller molecules and vesicles formed are approximately 100-150nm in diameter.

Figure 5

Caveolae-dependent endocytosis involves the regulation of many biological processes such as cell signalling, lipid regulation in the cellular membrane and diseases such as cancer. Caveolae are 50-80nm in size these vesicles that are created are known to bypass the lysozyme system and therefore are useful for gene and protein integration into the cell. ²⁸

Clathrin/caveolae independent endocytosis takes place in certain cells that have neither Clathrin or Caveolae proteins and use lipids such as cholesterol to ferry nutrients across the cellular membrane.²⁷

Macropinocytosis uses neither lipid rafts or proteins to transport nutrients into the cell instead of a protein as seen in figure ?? protrudes from the cell surface to cover the bulk fluid and draw it into the cell.²⁷

Figure 6 Macropinocytosis is another form of endocytosis that involves a plasma membrane protrusion that covers and pulls particulates into the cellular environment.²⁷

STO fibroblast cells taken from mice are a robust adherent cell line they are resistant to temperature and pH apoptosis making there perfect for live cellular imaging. Macrophage (Mice) cell lines are adherent but also able to undergo phagocytosis mediated endocytosis which is critical for nanoparticles above 200nm. As stated cubosomes have an approximate size range of 200-350nm depending on lipid makeup and processing this means phagocytosis and Clathrin-mediated endocytosis(CME) is the only viable endocytosis methods for our cell lines. ¹⁴

There is also evidence after an investigation into the effect of MO-based cubosomes on the cellular membrane A, Rosa et al.²⁹ It was found that MO cubosomes changed the intracellular membrane volume and reduced the cells lipid synthesis as well as saturating the cellular membrane.

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