Evidence-based approach for optimizing lipid-based nanosystems for

oral peptide delivery

Part I: solid lipid nanocarriers: development and characterization

Camille Dumont, PhD

May 2020
Presentation outline

Intestinal
Formulation
behavior
evaluation Conclusions and
Context and major take-home
issues messages

Caracterization
Context and major
issues
 Growing healthcare
Annual world market estimated at 13 billions $ (2016)  48-70 billions $ (2025)

Example: Leuprolide
500 peptides in pre-clinical development
30 - 90 days

140 peptides in clinical development Endometriosis

60 peptides approved by the Food and Drug Administration

oral
11 therapeutic forms for oral delivery on the market
 Encourage prescription earlier and more often
5 therapeutic forms for systemic absorption  Increase pharmaceutical compliance

 Quality of life improvement for thousands of women

Aguirre et al., Current status of selected oral peptide technologies in advanced preclinical development and in clinical trials,2016, Adv. Drug Del. Rev.
Richard, Challenges in oral peptide delivery: lessons learnt from the clinic and future prospects2017, Ther. Deliv.
Shields, Oral Peptide Therapeutics - A Holy Grail or Quixotic Quest?, 2017, Drug Disc. World
Fosgerau and Hoffman., Peptide therapeutics: current status and future directions, 2015, Drug Disc. Today
 DID YOU SAY PEPTIDE?

Small molecules Peptides Proteins

3-50 amino acids

500 Da 5000 Da
Molecular weight

 High molecular weight

 Generally hydrophilic

 Able to bind with specific receptors: ↗ capacity of action, ↘ adverse effects

 Natural elimination process: ↘ accumulation in tissues, ↘ toxicity
 Low half life in human physiological fluids

Morishita and Peppas., Is the oral route possible for peptide and protein drug delivery?, 2006, Drug Disc. Today
Craik et al., The Future of Peptide-based Drugs, 2013, Chem. Biol. Drug Des.
The GI tract, a series of barriers for peptides
Barrier n°1: Stomach
• Acidic environment
• Protease: pepsin

Barrier n°2: Duodenum, lumen

• Proteases: Trypsine, α-chymotryspine, elastase
• Glutathion (thiol/disulfide exchanges)

Barrier n°3: duodenum, intestinal border

• Mucus
• Hydrophobicity of epithelial cell plasmic membranes
• Low intercellular space / tight junctions

Low oral bioavailability (~1%)

Leonaviciute and Bernkop-Schnürch., Self-emulsifying drug delivery systems in oral (poly)peptide drug delivery 2015, Expert Opin. Drug Del.
Malhaire et al., How to design the surface of peptide-loaded nanoparticles for efficient oral bioavailability?, 2016, Adv. Drug Del. Rev.
Li et al., Oral delivery of peptides and proteins using lipid-based drug delivery systems, 2012, Expert Opin. Drug Deliv.
Advanced drug delivery systems: lipid nanocarriers

Solid

Liquid

Nanocarriers
Solid lipid
 Improved distribution nanocarrier
Nanoemulsion
 Increase permeability

Liquid

Self-Emulsifying Drug
Liposome
Lipids Delivery Sytem
 Biocompatibility and biodegradability
 ↗ transcellular transport
 Can act as permeation enhancers Nanocapsule

Almeida and Souto, Solid lipid nanoparticles as a drug delivery system for peptides and proteins, 2007, Adv. Drug. Deliv. Rev.
Mrsny, Oral drug delivery research in Europe, 2012, J. Controlled Release
Niu et al., Lipid-based nanocarriers for oral peptide delivery, 2016, Adv. Drug Deliv. Review
Solid lipid nanocarriers

Solid Lipid Nanoparticles, SLN Nanostructured Lipid Carriers, NLC

Surfactant Solid lipid Liquid lipid

Soares et al., Lipid nanocarriers loaded with natural compounds: Potential new therapies for age related neurodegenerative diseases?, 2018, Prog. In Neur.
Mehnert and Mäder, Solid Lipid Nanoparticles, Production, Characterization and Applications, 2012, Adv. Drug Deliv.
Müller et al., Solid lipid nanoparticles (SLN) for controlled drug delivery – a review of the state of the art, 2000, Eur. J. Pharm. Biopharm.
Beloqui et al., Nanostructured lipid carriers: Promising drug delivery systems for future clinics, 2016, Nanomed. Nanotechnol. Biol. Med.
Formulation
Peptide lipidization
Covalent lipidization:
• Reversible Aqueous Lipidization (REAL)
• Cyclization of the backbone
Formation of Hydrophobic H-bond Pair (HHP)

Ionizable amino acids at acidic pH pKa

Formation of Hydrophobic Ion Pair (HIP)
Arginine 13
Histidine 6
Lysine 11
Sodium Docusate
• Sulfonate group (pKa<0)
• Water solubility: 14 g/L, 25°C
Hydrophobic • Efficient HIP formation with more than 24 peptides and
Acidic medium anionic surfactant HIP proteins
Na+
Dumont et al., In-vitro evaluation of solid lipid nanoparticles: Ability to encapsulate, release and ensure effective protection of peptides in the gastrointestinal tract, 2019, Int. J. Pharm.
Nazir et al., Hydrophobic H-bond pairing: A novel approach to improve membrane permeability, 2019, Int. J. Pharm
Ristroph & Prud’homme., Hydrophobic ion pairing: encapsulating small molecules, peptides, and proteins into nanocarriers, 2019, Nanosc. Adv.
HIP formation with model peptides
Desmopressine Leuprolide
9 amino acids 8 amino acids
1069 g/mol 1209 g/mol
Log P = -2,5 Log P = -2

Optimal molar ratio peptide:docusate = 1:1,5 Optimal molar ratio peptide:docusate = 1:2

𝑃𝑒𝑝𝑡𝑖𝑑𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑎𝑓𝑡𝑒𝑟 𝐻𝐼𝑃

𝑃𝑟𝑒𝑐𝑖𝑝𝑖𝑡𝑎𝑡𝑖𝑜𝑛 𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 (𝐸𝑃, %) = 100 − × 100
𝑃𝑒𝑝𝑖𝑑𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑏𝑒𝑓𝑜𝑟𝑒 𝐻𝐼𝑃

Desmopressine: EP= 95,0 ± 0,3% (n=30)

Leuprolide: EP= 99,9 ± 0,1 % (n=118)
Griesser et al., Hydrophobic ion pairing: Key to highly payloaded self-emulsifying peptide drug delivery systems, 2017, Int. J. Pharm.
Formulation process selection

 Solvent-free process
 Ease of scale-up

Dumont et al., Lipid-based nanosuspensions for oral delivery of peptides, a critical review, 2018, Int. J. Pharm.
Dumont et al., A proof-of-concept for developing oral lipidized peptide Nanostructured Lipid Carrier formulations, 2019, J. Drug Del. Tech.
Solid lipid nanocarriers composition

Solid lipid excipient Liquid lipid excipient (NLC) Surfactant

5-10% 0.1-30% of lipidic fraction 2-5%

Excipient Mp (°C) Excipient HLB Excipient

Precirol®ATO5 50-60°C Capryol®90 5 Poloxamer 188

Maisine®CC 1 Poloxamer 407
Compritol®888 60-70°C
Labrafil® lipo WL1359 1 Polyoxyl-35 castor oil
Lauroglycol®90 1 Polyoxyl-40
hydrogenated castor oil
Peceol® 1
Polysorbate 80
Plurol oléique CC497 3

Mehnert and Mäder, Solid Lipid Nanoparticles, Production, Characterization and Applications, 2012, Adv. Drug Deliv.
Beloqui et al., Nanostructured lipid carriers: Promising drug delivery systems for future clinics, 2016, Nanomed. Nanotechnol. Biol. Med.
Formulation by High Pressure Homogenization
peptide +
liquid lipid
Ultrat
Ultraturrax
urrax

Melted
PATO5

400 rpm, 10 min 70°C 11000 rpm, 3 min 500 bar, 70°C 400 rpm, 10 min
70°C 70°C 5 cycles 4°C

Desmopressine Leuprolide

 No alteration of peptide during the process

Dumont et al., A proof-of-concept for developing oral lipidized peptide Nanostructured Lipid Carrier formulations, 2019, J. Drug Del. Tech.
Caracterization
EE depends on peptide log P
NLC composed of Precirol®ATO5 Capryol®90 Kolliphor® RH40
68% 5% 27%

100

80
EE (%)

60

40

20

0
EU
ES
-D

-L
IP
IP

-H
-H

LC
LC

N
N

Dumont et al., A proof-of-concept for developing oral lipidized peptide Nanostructured Lipid Carrier formulations, 2019, J. Drug Del. Tech.
Griesser et al., Hydrophobic ion pairing: Key to highly payloaded self-emulsifying peptide drug delivery systems, 2017, Int. J. Pharm.
Encapsulation efficiency
100%
*
90%
** **
80%
70%
60%

EE (%)
50%
40%
30%
20%
10%
0%

NLC LEU NLC HIP SLN LEU SLN HIP

Encapsulation efficiency of HIP or LEU as a function of the nanocarrier type

(* p<0.05, ** p<0.01) (Mean ± SEM, n=3)

 Very significant increase of the encapsulation efficiency when the peptide is lipidized

Dumont et al., In-vitro evaluation of solid lipid nanoparticles: Ability to encapsulate, release and ensure effective protection of peptides in the gastrointestinal tract, 2019, Int. J. Pharm.
Size characterization
Particle size measured by Blank NLC NLC-LEU NLC-HIP Blank SLN SLN-LEU SLN-HIP
Dynamic Light Scaterring Z-average (nm) 114 ± 11 113 ± 1 125 ± 2 119 ± 4 124 ± 1 127 ± 1
PDI 0.2 0.2 0.2 0.2 0.2 0.2

« nanospoons »

Edge-on particles

Top-view particles

Cryo-TEM images of: Blank NLC (A), NLC-LEU (B), NLC-HIP (C),
Blank SLN (D), SLN-LEU (E), SLN-HIP (F)
Dumont et al., In-vitro evaluation of solid lipid nanoparticles: Ability to encapsulate, release and ensure effective protection of peptides in the gastrointestinal tract, 2019, Int. J. Pharm.
Bunjes et al., Incorporation of the Model Drug Ubidecarenone into Solid Lipid Nanoparticles 2001, Pharm. Res.
Jores et al., Investigations on the structure of solid lipid nanoparticles (SLN) and oil-loaded solid lipid nanoparticles by photon correlation spectroscopy, field-flow fractionation and transmission
electron microscopy,2004, J. Contr. Rel.
Kuntsche et al., Cryogenic transmission electron microscopy (cryo-TEM) for studying the morphology of colloidal drug delivery systems, 2011, Int. J. Pharm
Intestinal behavior
evaluation
 Nanoparticles Stability in GI tract media
Simulated Gastric Fluid Fasted State Simulated Fed State Simulated
(SGF) Intestinal Fluid Intestinal Fluid
pH = 1.2 (FaSSIF-V2) (FeSSIF-V2)
pH = 6.5 pH = 5.8
Ionic strength ~100 mM ↗ Biliary salts
↗ Phospholipids
NLC SLN ↗ Fatty acids
Ionic strength ~200 mM
250 250

***
***

***
***
***
***
***
***

***
***

***
***

200 200

Mean diameter (nm)

***
**

***
***
Mean diameter (nm)

**
***

**
***
**
**

*
**

**

*
*

*
150 150

100 100

50 50

0 0
0 1 2 3 4 5 6 0 1 2 3 4 5 6
Time (h)
Time (h)
Water, 37°C SGF, 37°C FaSSIF-V2, 37°C FeSSIF, 37°C
Water, 37°C SGF, 37°C FaSSIF-V2, 37°C FeSSIF, 37°C

 No alteration of the nanosuspensions in the GI tract

Dumont et al., In-vitro evaluation of solid lipid nanoparticles: Ability to encapsulate, release and ensure effective protection of peptides in the gastrointestinal tract, 2019, Int. J. Pharm.
Protection against proteases
Trypsine α-chymotrypsine

• Protection
100% against proteases 100%

80% 80%
Leuprolide (%)

Leuprolide (%)
60% * 60%

**
40% 40%

20% 20%

0% 0%
0 50 100 150 0 10 20 30 40 50 60 70
Time (min) Time (min)

Degradation profile of Leuprolide in NLC LEU (), NLC HIP (), SLN LEU () and SLN HIP () by trypsine et l’α-chymotrypsine compared to free LEU () in
Tris-HCl pH 6,8 buffered medium (mean ± SEM, n=3, *p<0,05 et **p<0,01)

 Significant protection of LEU encapsulated in NLC attributed to non-solubility of trypsine in the liquid lipid excipients
 Degradation by α-chymotrypsine attributed to catalyctic effect of the nanoparticles on the proteases

Dumont et al., In-vitro evaluation of solid lipid nanoparticles: Ability to encapsulate, release and ensure effective protection of peptides in the gastrointestinal tract, 2019, Int. J. Pharm.
Hetényi et al., Comparison of the protective effect of self-emulsifying peptide drug delivery systems towards intestinal proteases and glutathione, 2017, Int. J. Pharm.
Intestinal transport – cell models

500 mL formulation diluted in HBSS

Caco-2
After 2h co-incubation monolayers are fixed and
HT29-MTX
stained to assess nanoparticles internalization

1 mL HBSS collected after 2h co-incubation with the formulation

to quantify the amount of transported peptide

Dumont et al., Lipid-based nanosuspensions for oral delivery of peptides, a critical review, 2018, Int. J. Pharm.
Solid nanoparticles internalization
Quantitative analysis: Quantitative anaysis:
Confocal Light Scanning Microscopy Fluorescence Activated Cell Sorting
SLN NLC
Caco-2

counts
Caco-2:HT29-MTX

% APC + /PE - Intensité de fluorescence

cellules moyenne (a.u.)

Contrôle 0.8 ± 0.1 32 ± 2

SLN 82.0 ± 4.6 1582 ± 438
NLC 98.8 ± 0.3 4558 ± 791

 Internalization of both nanocarriers

 Higher internalization for NLC compared to SLN
 Particles are able to cross mucus barrier
Article under review
Peptide transport across Caco-2 monolayer
2.9-fold increase in transport

Percentage of transport of exenatide from free solution and Exe-NLC, after co-incubation with the model for 2 h at 37 °C. Data shown as
mean ± SEM (N = 3, n = 12) (*p < 0.05, as compared to the control group) across the Caco-2 cell monolayer

 Solid lipid nanocarriers improve transport of peptides accross the intestinal barrier

Shrestha et al., The stimulation of GLP-1 secretion and delivery of GLP-1 agonists via nanostructured lipid carriers, 2018, Nanoscale
Promising in-vivo studies

Animal
Peptide Carrier Adminsitration Achievements Ref.
model
Diabetic (1)
Insulin SLN oral 5-fold bioavailabity increase compared to free insulin
rats
Salmon (2)
SLN Rats intragastrical Enhanced reduction of serum calcium levels (compared to free sCT)
calcitonin
Salmon • Increased bioalavailability (13%) (3)
SLN Rats intraduodenous
calcitonin • Increased pharmacological efficiency (17%)

In vivo studies indicate promising results combining peptides and solid lipid nanocarriers

(1) Ansari et al., Enhanced oral bioavailability of insulin-loaded solid lipid nanoparticles: pharmacokinetic bioavailability of insulin-loaded solid lipid nanoparticles in diabetic rats, 2016, Drug Deliv.
(2) Martins et al., Improving Oral Absorption of Samon Calcitonin by Trimyristin Lipid Nanoparticles, 2009, Jour. Biomed and Nanotech
(3) Chen et al., Orally delivered salmon calcitonin-loaded solid lipid nanoparticles prepared by micelle–double emulsion method via the combined use of different solid lipids, 2012, Nanomedicine
Conclusions
and
take-home
messages
Take-home messages
 Peptides can be encapsulated in SLN and NLC using HPH: a scalable and solvent-free process
 HPH enables formation of particles below 200 nm which favors intestinal transport

 Encapsulation in lipid nanocarriers depends on peptide log P : lipidization is generally mandatory

 Solid lipid nanocarriers are stable in the GI tract

 NLC protect peptides from enzymatic degradation induced by trypsine

 SLN and NLC are internalized by Caco-2 cells and are able to cross mucus

 Solid lipid nanocarriers increase peptide transport across intestinal barrier

 In-vivo studies report peptide bioavailability enhancement after encapsulation in SLN/NLC

Solid lipid nanocarriers are a promising system to increase oral bioavailability of peptides
Bibliography
Aguirre et al., Current status of selected oral peptide technologies in advanced preclinical development and in clinical trials,2016, Adv. Drug Del. Rev.
Almeida and Souto, Solid lipid nanoparticles as a drug delivery system for peptides and proteins, 2007, Adv. Drug. Deliv. Rev.
Beloqui et al., Nanostructured lipid carriers: Promising drug delivery systems for future clinics, 2016, Nanomed. Nanotechnol. Biol. Med.
Craik et al., The Future of Peptide-based Drugs, 2013, Chem. Biol. Drug Des.
Dumont et al., Lipid-based nanosuspensions for oral delivery of peptides, a critical review, 2018, Int. J. Pharm.
Dumont et al., In-vitro evaluation of solid lipid nanoparticles: Ability to encapsulate, release and ensure effective protection of peptides in the
gastrointestinal tract, 2019, Int. J. Pharm.
Dumont et al., A proof-of-concept for developing oral lipidized peptide Nanostructured Lipid Carrier formulations, 2019, J. Drug Del. Tech.
Fosgerau and Hoffman., Peptide therapeutics: current status and future directions, 2015, Drug Disc. Today
Griesser et al., Hydrophobic ion pairing: Key to highly payloaded self-emulsifying peptide drug delivery systems, 2017, Int. J. Pharm.
Hetényi et al., Comparison of the protective effect of self-emulsifying peptide drug delivery systems towards intestinal proteases and glutathione, 2017, Int. J. Pharm.
Jores et al., Investigations on the structure of solid lipid nanoparticles (SLN) and oil-loaded solid lipid nanoparticles by photon correlation spectroscopy,
field-flow fractionation and transmission electron microscopy,2004, J. Contr. Rel.
Kuntsche et al., Cryogenic transmission electron microscopy (cryo-TEM) for studying the morphology of colloidal drug delivery systems, 2011, Int. J. Pharm
Leonaviciute and Bernkop-Schnürch., Self-emulsifying drug delivery systems in oral (poly)peptide drug delivery 2015, Expert Opin. Drug Del.
Li et al., Oral delivery of peptides and proteins using lipid-based drug delivery systems, 2012, Expert Opin. Drug Deliv.
Malhaire et al., How to design the surface of peptide-loaded nanoparticles for efficient oral bioavailability?, 2016, Adv. Drug Del. Rev.
Mehnert and Mäder, Solid Lipid Nanoparticles, Production, Characterization and Applications, 2012, Adv. Drug Deliv.
Morishita and Peppas., Is the oral route possible for peptide and protein drug delivery?, 2006, Drug Disc. Today
Mrsny, Oral drug delivery research in Europe, 2012, J. Controlled Release
Müller et al., Solid lipid nanoparticles (SLN) for controlled drug delivery – a review of the state of the art, 2000, Eur. J. Pharm. Biopharm.
Nazir et al., Hydrophobic H-bond pairing: A novel approach to improve membrane permeability, 2019, Int. J. Pharm
Niu et al., Lipid-based nanocarriers for oral peptide delivery, 2016, Adv. Drug Deliv. Review
Richard, Challenges in oral peptide delivery: lessons learnt from the clinic and future prospects2017, Ther. Deliv.
Ristroph & Prud’homme., Hydrophobic ion pairing: encapsulating small molecules, peptides, and proteins into
nanocarriers, 2019, Nanosc. Adv.
Shields, Oral Peptide Therapeutics - A Holy Grail or Quixotic Quest?, 2017, Drug Disc. World
Soares et al., Lipid nanocarriers loaded with natural compounds: Potential new therapies for age related neurodegenerative diseases?, 2018, Prog. In Neur.

https://www.gattefosse.com/fr/pharmaceuticals-documentation/gattefosse-publications/
 Grateful acknowledgements to
• Gattefossé R&D
Dr Vincent Jannin
Cédric Miolane
• Université Claude Bernard Lyon 1:
Pr Hatem Fessi
Dr Sandrine Bourgeois
• Université Catholique de Louvain:
Pr Ana Beloqui
Pr Véronique Préat
• University of Innsbruck – Thiomatrix:
Pr Andreas Bernkop-Schnürch
Dr Jannine Griesser
Dr Gergely Hetényi
• European Network UNGAP
Thank you for your attention