Recent Advancement in Non-Invasive Delivery of Macromolecule Drugs

• St. Jones University

Macromolecules and Non-Invasive Delivery

Macromolecule drugs such as protein drugs have become a very important part of clinical treatments. There have been several hundred macromolecule drugs approved for clinical use. But mainly due to their low permeability through various absorption membranes, almost all of the macromolecule drugs are administered by injections with very few exceptions. Invasive or parenteral delivery of macromolecules comes with several limitations including systemic side effects, inconvenience to patients, and short duration of actions in many cases. There is a great need for a non-invasive delivery system for macromolecules. The non-invasive route is preferable as a convenient and easy way of administration with high patient compliance and flexibility in formulation. In addition, non-invasive dosage forms can significantly reduce the cost of clinical use because of self-administration of the drugs by the patients. The manufacturing cost may also be less for non-invasive dosage forms as compared to injections in many cases. ¹

Challenges and Advantages of Non-Invasive Delivery

The poor bioavailability from non-invasive delivery of macromolecules has been a major challenge. The physiochemical properties of macromolecules have a paramount effect on their bioavailability. Macromolecules are mainly of large molecular size and hydrophilic nature causing poor intrinsic permeation across biological membranes and low efficiency of cellular uptake. The inherent instability of macromolecules e.g. rapid degradation due to enzymes, pH and presence of water as well as fast clearance in most cases minimize the extent of macromolecule drugs reaching the site of action. Another barrier for macromolecule delivery is physiological factors. Different routes of administration consist of different conditions as shown in Table 1 either enhancing or compromising the bioavailability, thus presenting both opportunities and obstacles to research and development of non-invasive macromolecule drug delivery. ²

Pulmonary delivery is a promising route proven by the two insulin inhalation products approved by the FDA. The lungs possess a large surface area and an extensive vascular network, which are beneficial for absorption. However, the area of efficient absorption is in the alveolar region located in the deep lungs. Formulations with stable and proper aerodynamic diameter and certain surface properties are then required to pass through the multiple bifurcations of the air ducts to deposit in alveolar sac without being entrapped or cleared by mucociliary clearance in the airway and without any association in the warm and humid atmosphere of the airway. On the other hand, the deposited formulation in the alveolar sac may cause potential toxicity. Once deposited into the alveolar sac, the delivery system cannot be easily cleared although there is phagocytosis by macrophages in alveoli. But this phagocytosis is extremely limited and cannot be relied upon as the primary clearance mechanism for the drug delivery systems. ^2,3

Oral route is the most convenient route of administration with high patient compliance, but not the most efficient route for the delivery of macromolecules. The development of the oral macromolecule formulation requires overcoming pH variations and extensive proteolytic degradation existing from the oral cavity to the area of absorption in the GI tract. The permeability through the small intestine epithelium is another challenge even though it is relatively higher than other sites such as large intestine. ² The presence of Peyer’s patches may help the absorption of particulate delivery systems. But due to a limited number and small surface area of the patches, absorption through Peyer’s patch is very limited. The oral bioavailability of protein drugs is often further significantly reduced due to hepatic first-pass metabolism which is unique to oral route.

The intranasal route has great potential for macromolecule delivery. Interesting advantages are an abundance of vasculature structures, relatively high permeability and low enzymatic degradation activities. However, mucocilliary clearance causes the quick removal of the administered dose from the nasal cavity to the stomach. Another limitation is the relative small mucosal surface which limits the dose volume up to 200 - 300 μL per nostril. ² A unique benefit through intranasal route is the possibility to deliver the drugs to the brain through olfactory and trigeminal nerves.

The vaginal route is being explored as a new opportunity for non- invasive macromolecule delivery. Abundant vasculature structures and large surface area of vaginal expansion could benefit in the enhanced bioavailability of macromolecules. However, the menstrual cycles, age and gender inconsistently alter pH of the vaginal fluid, the composition of vaginal mucus, and the thickness of vaginal epithelium causing variations in the delivery of macromolecules. The irritation or toxicity of formulation in either short-term or long-term administration, cultural sensitivity, and personal hygiene thus become challenges. ⁴

Approaches For Non-Invasive Delivery

The non-invasive formulation strategies have been explored widely for macromolecule drugs. But so far, only very few have overcome the above-discussed challenges and become commercial products, and many are still in various development stages with some entering into clinical trials. Broad range of technologies have been used to overcome the barriers including increase of the drug lipophilicity, reduction of the drug molecular size, enhancement of the membrane permeability, prolongation of the retention time, and minimization of pre-absorption degradation. Current techniques applied are chemical modifications, absorption enhancers, particulate systems, lipid-based formulations, normal-flora, and enzyme inhibitors, providing some advantages and disadvantages as shown in Table 2.

Chemical Modifications

Chemical modification is used to modify macromolecule structures by conjugating with a chemical group or altering the backbone to improve lipophilicity, permeability, efficacy, and safety in both in vitro and in vivo stability. Ho et. al., performed PEGylation at the C-terminus of Nona-D-arginine (Poly R), called C-PEGylated Poly R, to enhance the vaginal uptake. ⁵ The vaginal delivery of C-PEGylated Poly Rs gel significantly increased the total serum concentration by 10-fold with 30k PEG and 3-fold with 1239k PEG as compared to parenteral in early pregnant mice. PEGylation increased the retention time of Poly R by its mucoadhesive properties facilitating the permeation across vaginal mucosa and prolonged half-life in terms of hepatic clearance reduction by small molecular weight PEG (30 k) or prevention of metabolism and renal clearance by high molecular weight PEG (1239k).

The study by Sheng et. al, found that low molecular weight protamine (LMWP) could be used as a cell penetrating peptide (CPP) for oral insulin delivery. ⁶ Both LMWP-linked insulin conjugate and insulin solution were loaded into mucoadhesive nanoparticles (MNPs) and delivered to diabetic rats over 12 hours. The result showed that the pharmacological availability, relative to subcutaneous injection of insulin solution, significantly improved from insulin solution to LMWP- linked insulin conjugate around 2-fold due to the higher permeability through intestinal mucosa, even though hypoglycemic effect between native insulin and insulin-LMWP conjugates without MNPs had no significant difference.

Absorption Enhancers

A variety of enhancers including bile salts, surfactants, Sodium N-[8-(2-hydroxybenzoyl) amino] caprylate (SNAC), sodium caprate etc. have been investigated and mainly proposed for opening the tight junction between the mucosal cells. ² Palmitoyl dimethyl ammoniopropanesulfonate (PPS), another absorption enhancer, was studied by Gupta et. al., for bovine insulin and salmon calcitonin (sCT) oral delivery. ⁷ Human colorectal adenocarcinoma Caco-2 cell line (HTB-37) was used for FITC-insulin with PPS permeation study and fasted adult male Sprague–Dawley (SD) rats were used for the efficacy study on sCT with PPS. The results showed that (1) increase of insulin permeation by 2.6 fold in presence of 0.03% w/v of PPS compared to insulin solution; (2) the presence of 1% PPS in sCT solution significantly helped in reducing the plasma calcium as much as 56 ± 2% from the initial values, and increased AUC by 49- fold as compared to sCT solution alone; (3) the penetration ability of PPS was concentration-dependent: 0.03% of PPS resulted in 2-fold higher apparent permeability and percentage transport of sCT than 0.01% PPS. This approach is not limited for oral delivery. Chitosan is another absorption enhancer, which has been explored for several routes of administration. The study of Plessis et. al., on N-trimethyl chitosan chloride (TMC) in intranasal delivery of sCT focused on the properties of TMC as an absorption enhancer. The in-vivo study on rats showed that the presence of 0.5% w/v TMC in 100 IU/ml sCT solution increases the sCT serum concentration approximately 7-fold through intranasal delivery. ⁸

Particulate Systems

Polymeric particulate systems commonly known as liposomes, encapsulations, micro- or nano- particles provide the advantages like protection from degradation in acidic environment, delivery to absorption site, prolonged retention time, etc. Alibolandi et. al., used Dextran-PLGA for insulin encapsulation to form polymersomes (DEX-PL). ⁹ Insulin-loaded DEX5000-PLGA13000 with 139.2 ± 12.23 nm particle size were delivered to diabetic rats orally. Significant hypoglycemic effect was observed with 9.77% bioavailability as compared to oral insulin solution with 0.62% bioavailability.

Particulate systems have been successfully developed for pulmonary delivery of macromolecules. Two insulin inhalation powders were approved by FDA in 2006 and 2014, respectively. In the late insulin inhalation product (Afrezza), fumaryl diketopiperazine is used as the major component to form a particle matrix of an average particle size of 2.5 μm as a carrier for insulin. This product showed similar bioavailability as s.c. injection of insulin solution. A clinical trial on Type-II diabetic patients indicated that 37.7% of patients showed HbA1c glycated haemoglobin less than 7% by this product, which was significantly higher than placebo group. The clinical trial on Type-I diabetic patients also showed the significant achievement: (1) an average 0.4 kg weight lost as compare to 0.9 kg weight gain by s.c. insulin solution; (2) Fasting plasma glucose (FPG) reduction of 25.3 mg/dL significantly greater than s.c. insulin solution. However, the number of patients with HbA1c lower than 7% was greater in the group of s.c. insulin solution. ^10,11

Lipid-Based Formulations

Lipid based drug delivery has emerged as a promising delivery approach for some drugs, especially for the small water-insoluble drugs and hydrophilic macromolecules. ^12,13 O/W nanoemulsions formed by self-nano emulsifying drug delivery systems (SNEDDS) offer several advantages like long-term stability, consistency in droplet size, high reproducibility, and ease of preparation. ^14,15 The macromolecule can be loaded in the lipid phase of O/W nanoemulsion via solid dispersion technique. ¹⁶

One of the interesting study on SNEDDS for oral insulin delivery has been reported by Li et. al. Soy bean phospholipid was used to form a complex with insulin and then loaded inside the SNEDDS at different concentration (2.5% or 10%). Then, the insulin-loaded SNEDDS was filled in Eudragit®L100 capsules which were administered orally to fasted rats by gavage and compared with control groups – insulin in phosphate buffer solution, and 2.5% and 10% complex-loaded in uncoated capsule. The percentage pharmacological activity after three hours of the complex-loaded in enteric coated capsules were significantly higher than insulin solution at p < 0.01 with the maximum glucose reduction of 38%. However, the uncoated capsules did not result in this kind of significant difference. ¹⁷

Normal Flora Engineering

Normal flora modified by recombinant DNA technology can secrete selected peptide/protein drugs. Normal flora can adhere tightly to epithelial cell surfaces and thus are able to deliver the drug directly on the absorption epithelium. The study of recombinant L. lactis with plasmid pUBGLP-1 by Agarwal, P. et al showed the secretion of GLP-1 at the site of absorption i.e. intestine epithelium and when given orally to Zucker diabetic rats, lowered plasma glucose level by 10-20% and increased serum insulin by 2.5-fold. ⁶ Another study from Kaushal and Shao on gene transformed L. lactis for beta-lactamase (29 kDa) vaginal delivery in rats showed a significant improvement in absorption of beta-lactamase compared to the solution form. The absorption result was dose-dependent, when the dose increased from 1.2 x 10 ⁷ to 3.0 x 10 ⁷ colony forming units, approximately 2.5-fold of AUC was presented. With the increased dose, the mean residence time and mean absorption time were prolonged from 15 hr to 20 hr and from 10 hr to 15 hr, respectively, demonstrating the benefit of normal flora to prolong the delivery. ¹⁸

Enzyme Inhibitors

Enzyme inhibitors may be delivered together with macromolecules to avoid the enzymatic degradation such as DPP-4 inhibitor for GLP- 1, trypsin and α-chymotrypsin inhibitors for insulin. Oral exenatide formulation composed of two inhibitors – Soybean Trypsin Inhibitor (SBTI) and Na-EDTA has been patented and is currently in clinical Phase II trial. The inhibitors together with omega-3 fatty acid are used to facilitate the intestine absorption. The protease inhibition capability was tested in beagle dogs and pigs by administration with exenatide 50 μg to duodenum, which resulted in the protection of exenatide from the enzymatic degradation. ¹⁹

Conclusion

Non-invasive delivery of macromolecules has remained a difficult challenge over years. Until now, only several relatively small molecules such as insulin (~5 kDa) have been successfully formulated into such a delivery system. Different sites of administration present different obstacles and advantages, and an effective formulation is highly compound specific. In general, integration of various approaches is required to improve the non-invasive delivery of macromolecule drugs to reach a satisfactory therapeutic potential.

References

1. Van Der Walle CF, ed. Peptide and protein delivery. first ed. Elsevier; 2011.
2. Shao J, Khatri P, Savla R, Lehr C, Stamoran c, eds. Non-invasive macromolecule delivery guide. Catalent Institute Press; 2015.
3. Kunda NK, Alfagih IM, Dennison SR, et al. Dry powder pulmonary delivery of cationic PGA-co-PDL nanoparticles with surface adsorbed model protein. Int J Pharm. 2015;492(1):213-222.
4. Machado RM, Palmeira-De-Oliveira A, Martinez-De-Oliveira J, Palmeira-De-Oliveira R. Vaginal films for drug delivery. J Pharm Sci. 2013;102(7):2069-2081.
5. Ho H, Nero TL, Singh H, Parker MW, Nie G. PEGylation of a proprotein convertase peptide inhibitor for vaginal route of drug delivery: In vitro bioactivity, stability and in vivo pharmacokinetics. Peptides. 2012;38(2):266-274.
6. Agarwal P, Khatri P, Billack B, Low W, Shao J. Oral delivery of glucagon like peptide-1 by a recombinant lactococcus lactis. Pharm Res. 2014;31(12):3404-3414.
7. Gupta V, Hwang BH, Doshi N, Mitragotri S. A permeation enhancer for increasing transport of therapeutic macromolecules across the intestine. J Controlled Release. 2013;172(2):541-549.
8. Plessis L, Lubbe J, Strausset T. Enhancement of nasal and intestinal calcitonin delivery by the novel pheroid TM fatty acid based delivery system and by N-trimethyl chitosan chloride. Int J Pharm. 2010;385(1-2):181-186.
9. Alibolandi M, Alabdollah F, Sadeghi F, et al. Dextran- b-poly (lactide-co-glycolide) polymersome for oral delivery of insulin: In vitro and in vivo evaluation. J Controlled Release. 2016; 227:58-70.
10. Boss AH, Petrucci R, Lorber D. Coverage of prandial insulin requirements by means of an ultra-rapid- acting inhaled insulin. J Diabetes Sci Technol. 2012;6(4):773-779.
11. Kim ES, Plosker GL. AFREZZA®(insulin human) inhalation powder: A review in diabetes mellitus. Drugs. 2015;75(14):1679-1686.
12. Dahan A, Hoffman A. Rationalizing the selection of oral lipid based drug delivery systems by an in vitro dynamic lipolysis model for improved oral bioavailability of poorly water soluble drugs. J Controlled Release. 2008;129(1):1-10.
13. Hintzen F, Perera G, Hauptstein S, Müller C, Laffleur F, Bernkop-Schnürch A. In vivo evaluation of an oral self- microemulsifying drug delivery system (SMEDDS) for leuprorelin. Int J Pharm. 2014;472(1):20-26.
14. Mu H, Holm R, Müllertz A. Lipid-based formulations for oral administration of poorly water-soluble drugs. Int J Pharm. 2013;453(1):215-224.
15. Djekic L, Primorac M. The influence of cosurfactants and oils on the formation of pharmaceutical microemulsions based on PEG-8 caprylic/capric glycerides. Int J Pharm. 2008;352(1):231-239.
16. Rao SVR, Yajurvedi K, Shao J. Self-nanoemulsifying drug delivery system (SNEDDS) for oral delivery of protein drugs: III. in vivo oral absorption study. Int J Pharm. 2008;362(1):16-19.
17. Li P, Tan A, Prestidge CA, Nielsen HM, Müllertz A. Self-nanoemulsifying drug delivery systems for oral insulin delivery: In vitro and in vivo evaluations of enteric coating and drug loading. Int J Pharm. 2014;477(1):390-398.
18. Kaushal G, Shao J. Vaginal delivery of protein drugs in rats by gene-transformed lactococcus lactis. Drug discoveries & therapeutics. 2009;3(5).
19. Kidron M, inventorMethods and compositions for oral administration of exenatide. patent 12/990,097. 2009.