Stuart R. Gallant, MD, PhD

Proteins and peptides are commonly delivered subcutaneously and by infusion; however, these approaches have disadvantages.  Among the chief problems are:  discomfort at the injection site, inconvenience of administration at a medical office or infusion center, and added expense of an autoinjector or other administration supplies.

For protein drugs, the dream is to administer protein and peptide drugs via tablet or capsule.  Today’s post looks at technology for oral administration of proteins and peptides.  The array of oral delivery systems for protein drugs is truly amazing:

Given the many diverse strategies, this post is not able to address all of them.  Instead, the goal of the post is to look at a few key aspects of gut anatomy and then move on to discuss some important aspects of formulation, particle delivery, receptor targeting, and sublingual delivery.  The reader is reminded of an earlier post on PharmaTopo that contains some useful information about oral dosage forms [1].

The Gut

There are several strong barriers to oral delivery of protein and peptide drugs.  The first roadblocks are contained in the contents of the stomach and the small and large intestines:

• Protein drugs are denatured and degraded by two significant features of the gastrointestinal tract:  1) hydrochloric acid released by the parietal cells of the stomach and 2) pancreatic digestive enzymes (trypsinogen, chymotrypsinogen, elastase, and carboxypeptidase).
• The small intestine and particularly the colon are not sterile.  Bacteria present in the gastrointestinal tract secrete their own digestive enzymes.

Most absorptive activity in the gastrointestinal tract occurs in the small intestine.  The 3-to-5-meter length of the small intestine provides substantial surface area which is in turn multiplied many fold because of the presence of intestinal villi, tiny (0.5 to 1.5 mm long) fingers of epithelial cells which project into the lumen of the small intestine [2]:

The surface of the villi is composed of enterocytes (as well as goblet cells which are no shown in the figure above).  The enterocytes start their lifecycle in the crypts of the villi where stem cells constantly renew the population of enterocytes.  Above the crypts, along the sides of the villi are rapidly dividing enterocytes (with a cell cycle of about 12 hours).  At the tips of the villi, nondividing, fully differentiated enterocytes.  These cells are eventually sloughed into the lumen of the intestine where they eventually leave the body in feces.

In addition to the enterocytes, there are goblet cells interspersed along the surfaces of the villi.  Goblet cells secret a layer of mucus which coats the surface of the villi.  A few goblet cells are indicated by black arrows in the figure below:

Within the villi are arteries and veins supplying the metabolic needs of the adsorptive and mucus secreting cells.  Once adsorbed molecules have made it past the barrier formed by the enterocytes, they can pass into the venous circulation, leading to the mesenteric veins which join the portal vein.  The portal vein supplies the liver where first pass metabolism occurs before blood returns to the heart and lungs.

What roadblocks and toll gates exist for absorption from the intestinal lumen to the venous circulation?

• The enterocytes form tight junctions with their neighboring cells.  The tight junctions have net negative charge and small pore size (8-13Å).  So, any molecule carrying a charge or with characteristic size much larger than glucose will find the tight junctions impassible.
• Lipophilic molecules can pass through the enterocyte cell membrane and emerge on the internal side of the cellular membrane.  The mechanism is simple diffusion.
• In transcytosis, a vesicle forms on the lumen side of an enterocyte [2].  This may be mediated by a receptor or it not.  The vesicle may interact with endosomes within the cell, and eventually emerges on the opposite side of the enterocyte at the basal side of the cell where the contents of the vesicle are delivered to the extracellular fluid.
• Efflux pumps appear on the lumen side of enterocytes, returning some molecules which have gained entry to the cytosol back into the lumen of the intestine.
• Microfold cells or M cells:  M cells are cells of the immune system located in collections of cells known as Peyer’s patches within the small intestine.  M cells are particularly avid in their endocytosis activities.  For instance, it is thought that they are particularly responsible for uptake of nanoparticulate drugs.  As result, they represent a significant possible gateway from the lumen of the small intestine to venous circulation.

The epithelial cells lining the gut constitute a primary protective barrier to a person, preventing loss of interstitial fluid and guarding against intrusion of bacteria, viruses, and toxins.  Oral delivery of protein drugs employs agents that could conceivably compromise this important protective layer.  Any dosage strategy using these types of technologies should take great care that any manipulation of the protective properties of the gut is:  1) temporary and followed by rapid restoration of the normal behavior of the gut and 2) insubstantial and does not allow infection or intoxication to occur.  These two criteria (temporariness and insubstantiality) should apply to patients with normal gut behavior and to patients with impaired gut behavior (e.g., Crohn’s disease, ulcerative colitis, celiac disease, and inflammatory bowel disease).

Protective Agents

Having considered the array of defenses that prevent absorption of therapeutic proteins, what formulation strategies exist to stabilize and protect proteins in the gastrointestinal tract and to enhance absorption?

• Capsules and Coated Tablets:  Both capsules and coated tablets can provide protection against the acid environment of the stomach [1].  Tablet formation can generate significant stress on the drug product components due to the pressure involved.  This stress can be sufficient to denature protein components.  Each product is different; however, encapsulation is likely the preferred technology for protection of protein products against the environment of the stomach (i.e., compared to tablet formation followed by enteric coating).
• Coated Particles:  Similar to capsules, coated particles provide protection to the active pharmaceutical ingredient contained within them.  Coated particles maybe provided to the patient as encapsulated products or as stick packs.  Manufacture of micro or nanoparticles is possible as laboratory scale and at commercial scale.  The choice of micro or nano scale depends on dissolution rates for the particular polymer system employed.  Three possible manufacturing methods for poly (D, L-lactic-co-glycolic acid) (PLGA) are shown below [3]:

• Formulation Agents for Protein Stability:  Formulation agents used to stabilize protein drug products during storage include [4]:
  • Buffers:  Phosphate, histidine and other buffer species prevent low pH degradation of proteins.
  • Sugars:  Mannitol and other sugars protect proteins, particularly during storage by freezing.
  • Surfactants:  Polysorbates and other surfactants inhibit aggregation.
  • Salts:  Sodium chloride and other salts also inhibit aggregation.
• Formulation Agents to Enhance Absorption:  Formulation agents used to enhance absorption include:
  • Protease inhibitors:  Protease inhibitors have the potential to halt degradation long enough for drug adsorption to occur within the intestine; however, the large dilution that occurs in the intestine creates two problems for this strategy:  1) To be successful, a large bolus of protease inhibitor would need to be included in the drug product capsule—greatly increasing the size of the dosage form.  Alternatively, two capsules (one with the active and one with the protease inhibitor) must be ingested at the same time.  2) At these high protease inhibitor doses, the action of the protease inhibitor may cause side effects [5].
  • Chelating agents:  Ethylenediaminetetraacetic acid (EDTA) avidly binds to calcium, reducing the effectiveness of the tight junctions between enterocytes [6].  The result is enhanced paracellular transport.
  • Bile salts and detergents:  Solubility enhancers such as bile salts and detergents can improve transport across the enterocyte barrier [7].  One mechanism for improved adsorption is enhanced paracellular transport, again by reducing the effectiveness of the tight junctions between enterocytes.

Particulate Delivery Systems to the Intestine

A widely studied protein as a candidate for oral delivery is insulin.  It’s a fairly robust small protein (51 amino acids, 5808 Da) for which a large market would be available if the correct oral delivery technology could be identified.  Here are some examples of particulate technologies which have been tested for delivery of insulin:

• Dextran Sulfate/Chitosan Nanoparticles:  Nanoparticles offer protection to protein drugs.  In addition, they enhance absorption by lengthening the transit time through the gut due to entrapment in the intestinal mucus layer and by being taken up by M cells of the Peyer’s patches in the small intestine.  Sarmento and colleagues investigated insulin delivery using insulin-loaded dextran sulfate/chitosan nanoparticles [8].  In diabetic rats, the bioavailability was:  oral solution (control condition) at 50 IU/kg—1.6%, nanoparticles at 50 IU/kg—5.6%, nanoparticles at 100 IU/kg—3.4%.

We can see several lessons from Sarmento’s work.  First, despite the barriers to protein absorption, some insulin does reach central circulation even in the least favorable situation (delivery by oral solution).  Second, protecting insulin within nanoparticles boosts bioavailability by a factor of 3.5x.  Some other particle technologies offer similar insulin delivery performance to dextran sulfate/chitosan nanoparticles.  These include:

• Solid Lipid Nanoparticles (SLNs):  In diabetic rats, Ansari and coworkers were able to acheive a bioavailability of 8.3% using solid lipid nanoparticles prepared using a water in oil in water double emulsion–solvent evaporation technique (see simplified process flow diagram above) [9].
• LDH-DCA-HA:  Huang and coworkers investigated layered double hydroxide (LDH) nanoparticle modified by deoxycholic acid (DCA) and hyaluronic acid (HA) [10].  They did not collect bioavailability data, but they were able to improve transport through a Caco-2 monolayer from 4.7 x 10^-7 cm/s for insulin solution to 10.9 x 10^-7 cm/s for LDH-DCA-HA nanoparticles—an increase of 2.3x in rate of diffusion.  The authors theorize that the formulation agents reversibly opened the tight junctions between the CaCo-2 cells.

The real lottery ticket winner for delivery of insulin is mesoporous silica nanoparticles:

• Mesoporous Silica Nanoparticles (MSNs):  Guha and coworkers synthesized PMV [poly (methacrylic acidco-vinyl triethoxylsilane)]-coated mesoporous silica nanoparticles (MSNs) [11].  MSNs by a process summarized in the figure below.  With MSNs, a bioavailability of 73% in rabbits was achieved.

Having found a delivery system that provides insulin orally with a high bioavailability, what do the pharmacokinetics look like?  Comparing subcutaneous delivery (dashed line) to oral administration (solid line) in rabbits, the plasma insulin level is substantially more sustained in the oral administration:

This sustained level would perhaps be appropriate for “basal” insulin [12].  Unfortunately, the peak is too broad for mealtime insulin.  However, for many protein drugs, such a sustained release might be acceptable or even beneficial.

Targeting Intestinal Receptors

Receptor mediated endocytosis may be the first step to transcytosis (see discussion above).  One way of significantly boosting transcytosis rates is targeting right receptor, and one way to target a receptor is by creation of a fusion protein with one portion being the active drug and the other portion being a protein which interacts with a target on the enterocyte surface.  An example of this strategy creates a fusion protein of IL-10 with a nontoxic fragment of cholera toxin [13].  The resulting protein is known as AMT-101 (125 kDa).  The toxin fragment of AMT-101 binds to GM1 gangliosides on the enterocyte surface, permitting transcytosis of the fusion protein.

Fay and colleagues do not report a bioavailability.  They do report a plasma level of IL-10 of 700 pM at 4 hours for a dose of 10 mg/kg.  With some modest assumptions (25 gram mice, 2 ml blood volume), that corresponds to 0.07% of the dose present in the plasm at 4 hours.  This correlates with a relatively modest bioavailability.

Sublingual Delivery

Deamino D-arginine vasopressin (DDAVP, 1183 Da) is a synthetic analog of the endogenous human hormone, 8-arginine vasopressin (aka, antidiuretic hormone):

In a study by Fjellestad-Paulsen and coworkers, a range of routes of administration were compared and urinary DDAVP was measured:

	Dose (micrograms)	24 Hr Excretion (pmol)	24 Hr Excretion (micrograms)	Excreted (%)
Intravenous	2	738	0.87	43.7%
Subcutaneous	2	902	1.07	53.4%
Intranasal	20	599	0.71	3.5%
Oral	200	122	0.14	0.1%
Sublingual	20	16	0.02	0.1%
Intrarectal	50	22	0.03	0.1%

In health subjects, about 50% of DDVAP is cleared renally.  Using intravenous administration as the reference point, subcutaneous administration is roughly equally bioavailable (with an excreted percent close to 50%).  The oral doses varied in strength because it was expected that the non-parenteral doses would be less bioavailable.  Intranasal had less than 3.5% excretion (bioavailability of about 7%).  Oral, sublingual, and intrarectal, all had bioavailability of about 0.2%.

This low sublingual bioavailability contrasts with insulin bioavailability when delivered by sublingual microemulsion [14].  The microemulsion contained a surfactant (Tween 80), co-surfactant (diethyleneglycol monoethyl ether/Soluphor P) and an oil (caprylic acid), along with permeation enhancers noted in the table below:

Formulation	Route	Bioavailability
Subcutaneous (Control)	SC	100%
Microemulsion with Transcutol	SL	52%
Microemulsion with Transcutol with TPGS	SL	62%
Microemulsion with Transcutol with Aerosol OT	SL	55%
Microemulsion with Transcutol with Nicotinamide	SL	43%
Microemulsion with Soluphor P	SL	25%

As can be seen in the table, with the correct formulation, it is possible to achieve significant sublingual absorption of proteins.

Conclusions

The problem of oral delivery of protein drug products is a complex one that includes:

• Active ingredient stability during storage
• Bioavailability in the context of the delivery technology
• Side effects and toxicities of the delivery technology

This last point is often overlooked, but nevertheless important.  The body has a virtually infinite number of locations to deliver a subcutaneous dose, so rotation of injection site is quite easy.  In contrast, there is only one underside of the tongue.  So, if any irritation develops as a result of sublingual delivery, that creates a significant challenge to that route.  There are methods of overcoming such challenges, but they require thought in advance.

This post has hopefully shown that, while complex, oral delivery of proteins drugs is not an insoluble problem and one worth considering during preclinical development.

[1] Gallant, S.R.  “Immediate, Delayed, and Extended-Release Drug Products,” PharmaTopo.com, November 14 (2022).  pharmatopo.com/index.php/2022/11/14/immediate-delayed-and-extended-release-drug-products/

[2] Alberts, B., et al.  Molecular Biology of the Cell, 6^th Edition.

[3] Ding, D. and Zhu, Q.  “Recent advances of PLGA micro/nanoparticles for the delivery of biomacromolecular therapeutics,” Mater Sci Eng C Mater Biol Appl 1;92:1041-1060 (2018).

[4] Gallant, S.R.  Formulation Resources, www.sandpiperpharma.com/formulation/

[5] Smith, J.C., et al.  “Hypertrophy and hyperplasia of the rat pancreas produced by short-term dietary administration of soya-derived protein and soybean trypsin inhibitor,” J Appl Toxicol 9(3):175-9 (1989).

[6] Tomita, M., et al.  “Absorption-Enhancing Mechanism of EDTA, Caprate, and Decanoylcarnitine in Caco-2 Cells,” J Pharm Sci 85(6):608-11 (1996).

[7] Moghimipour, E., et al.  “Absorption-Enhancing Effects of Bile Salts,” Molecules 20(8):14451-73 (2015).

[8] Sarmento, B., et al.  “Oral Bioavailability of Insulin Contained in Polysaccharide Nanoparticles,” Biomacromolecules 8, 3054-3060 (2007).

[9] Ansari, M.J., et al.  “Enhanced oral bioavailability of insulin-loaded solid lipid nanoparticles: pharmacokinetic bioavailability of insulin-loaded solid lipid nanoparticles in diabetic rats,” Drug Deliv 23(6):1972-9 (2016).

[10] Huang, X., et al.  “Layered Double Hydroxide Modified with Deoxycholic and Hyaluronic Acids for Efficient Oral Insulin Absorption,” Int J Nanomedicine 1;16:7861-7873 (2021).

[11] Guha, A., et al.  “pH responsive cylindrical MSN for oral delivery of insulin-design, fabrication and evaluation,” Drug Deliv 23(9):3552-3561 (2016).

[12] Gallant, S.R.  “Pharmaceutical Design Focus:  Insulin,” PharmaTopo.com, February 5 (2022).  pharmatopo.com/index.php/2022/02/05/pharmaceutical-design-focus-insulin/

[13] Fay, N.C., et al.  “A Novel Fusion of IL-10 Engineered to Traffic across Intestinal Epithelium to Treat Colitis,” J Immunol 205 (11): 3191–3204(2020).

[14] Patil, N.H. and Devarajan, P.V.  “Enhanced insulin absorption from sublingual microemulsions: effect of permeation enhancers,” Drug Deliv. and Transl. Res. 4:429–438 (2014).

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