IE19980809A1 - Composition for enhancing transport across gastrointestinal tract cell layers - Google Patents
Composition for enhancing transport across gastrointestinal tract cell layersInfo
- Publication number
- IE19980809A1 IE19980809A1 IE1998/0809A IE980809A IE19980809A1 IE 19980809 A1 IE19980809 A1 IE 19980809A1 IE 1998/0809 A IE1998/0809 A IE 1998/0809A IE 980809 A IE980809 A IE 980809A IE 19980809 A1 IE19980809 A1 IE 19980809A1
- Authority
- IE
- Ireland
- Prior art keywords
- oil
- monolayers
- trh
- babassu
- teer
- Prior art date
Links
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- FDJOLVPMNUYSCM-WZHZPDAFSA-L cobalt(3+);[(2R,3S,4R,5S)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2R)-1-[3-[(1R,2R,3R,4Z,7S,9Z,12S,13S,14Z,17S,18S,19R)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+3].N#[C-].N([C@@H]([C@]1(C)[N-]\C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C(\C)/C1=N/C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C\C1=N\C([C@H](C1(C)C)CCC(N)=O)=C/1C)[C@@H]2CC(N)=O)=C\1[C@]2(C)CCC(=O)NC[C@@H](C)OP([O-])(=O)O[C@H]1[C@@H](O)[C@@H](N2C3=CC(C)=C(C)C=C3N=C2)O[C@@H]1CO FDJOLVPMNUYSCM-WZHZPDAFSA-L 0.000 description 1
- 239000003240 coconut oil Substances 0.000 description 1
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- 201000010897 colon adenocarcinoma Diseases 0.000 description 1
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- 238000006731 degradation reaction Methods 0.000 description 1
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- 239000000975 dye Substances 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 238000007046 ethoxylation reaction Methods 0.000 description 1
- 230000003203 everyday Effects 0.000 description 1
- 231100000910 evident toxicity Toxicity 0.000 description 1
- 150000002191 fatty alcohols Chemical class 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 230000002496 gastric Effects 0.000 description 1
- 125000005456 glyceride group Chemical group 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- 229940020899 hematological Enzymes Drugs 0.000 description 1
- 230000002440 hepatic Effects 0.000 description 1
- 230000003301 hydrolyzing Effects 0.000 description 1
- 230000002267 hypothalamic Effects 0.000 description 1
- 230000002989 hypothyroidism Effects 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 229940119170 jojoba wax Drugs 0.000 description 1
- 239000004951 kermel Substances 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 229950008325 levothyroxine Drugs 0.000 description 1
- 230000000670 limiting Effects 0.000 description 1
- 239000000944 linseed oil Substances 0.000 description 1
- 235000021388 linseed oil Nutrition 0.000 description 1
- 229950004592 liothyronine Drugs 0.000 description 1
- 230000002132 lysosomal Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000035786 metabolism Effects 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 230000002438 mitochondrial Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000006011 modification reaction Methods 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 239000008164 mustard oil Substances 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 230000001452 natriuretic Effects 0.000 description 1
- 201000002652 newborn respiratory distress syndrome Diseases 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 230000003000 nontoxic Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- IAYPIBMASNFSPL-UHFFFAOYSA-N oxane Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 description 1
- 101700057139 oxyT Proteins 0.000 description 1
- 239000000312 peanut oil Substances 0.000 description 1
- 239000003961 penetration enhancing agent Substances 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 239000004014 plasticizer Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000002335 preservative Effects 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 239000001044 red dye Substances 0.000 description 1
- 230000001105 regulatory Effects 0.000 description 1
- 239000003340 retarding agent Substances 0.000 description 1
- 230000002441 reversible Effects 0.000 description 1
- 235000005713 safflower oil Nutrition 0.000 description 1
- 239000003813 safflower oil Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000008159 sesame oil Substances 0.000 description 1
- 235000011803 sesame oil Nutrition 0.000 description 1
- 231100000486 side effect Toxicity 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- FIWQZURFGYXCEO-UHFFFAOYSA-M sodium;decanoate Chemical compound [Na+].CCCCCCCCCC([O-])=O FIWQZURFGYXCEO-UHFFFAOYSA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- NHXLMOGPVYXJNR-ATOGVRKGSA-N somatostatin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CSSC[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N1)[C@@H](C)O)NC(=O)CNC(=O)[C@H](C)N)C(O)=O)=O)[C@H](O)C)C1=CC=CC=C1 NHXLMOGPVYXJNR-ATOGVRKGSA-N 0.000 description 1
- 229960004532 somatropin Drugs 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
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- 238000007619 statistical method Methods 0.000 description 1
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- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 238000003211 trypan blue cell staining Methods 0.000 description 1
- 229960001322 trypsin Drugs 0.000 description 1
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- 235000013311 vegetables Nutrition 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
-
- Y10S514/80—
-
- Y10S514/802—
-
- Y10S514/807—
-
- Y10S514/808—
-
- Y10S514/822—
Abstract
Abstract A composition for enteral administration having a non-ionic vegetable oil GlT absorption enhancer capable of increasing the enteral absorbability of drugs, especially oral absorbability of hydrophilic and macromolecular drugs. The non- ionic vegetable oil GIT absorption enhancer, particularly Babassu oil or a derivative thereof, is capable of enhancing the uptake of a drug from the gastrointestinal tract so as to allow therapeutically effective amounts of the drug to be transported across the GlT of an animal such as a human without significant toxic side effects. 1 /7 IE980809 INFORIVIA L DRAWIN GS 100 ‘ K U) T . H ‘:5 } , E 80 {U ‘\_ 5 60 %% 05 . 5 + 1.14 8 20 ' at I 38 0 ' ‘ ‘u a H .:’1'* 60 EU Time (minutes) Fig. 1 % Resistance Remaining 2,? IE980809 INFORMAL DRAWINGS L 36 66 90 Time (minutes) Fig. 2 °/o Resistance Remaining 100 « 30 ~ INFORMA L DRAWINGS Time (minutes) Fig. 3 IE980809 j—C?< 4;7 IE980809 INFORMA L DRAWIN GS 100 «r L. U) CD CD CD I-—,+--I .—o.— " 3' E v——0——-c >--—O—---I r—6—-4 % Resistance Remaining r: 30 60 90 «Aw Time (minutes) Fig. 4 IE980809 INFORMAL DRAWINGS 100 T f O3 on C3 O -—-O— 9--'0--or €»—a+- % Resistance Remaining U 1:13? 60 ml‘) 1;?!) Time (minutes) Fig. 5 "/3 Resistance remaining 6.’? INFORMAL DRAWINGS men‘ --o-+_.s-Q fie...‘ ‘ ) , U Iii I 60 90 Time (minutes) Fig. 6 IE980809 \{ H470 -.-._-PK70 (M70 +43A7OG % Resistance Remaining 100 4,. ,3 INFORMAL DRAWIN GS IE980809 . A70 _._.PK70 .. M70 . BA"/’0G
Description
COMPOSITION FOR ENHANCING TRANSPORT ACROSS
GASTROINTESTINAL TRACT CELL LAYERS
The present invention relates to a composition and its use for
enhancing transport across gastrointestinal tract (GIT) cell layers in an
animal.
While the vast majority of drugs are intended for systemic action,
drug formulations are designed for enteral or peroral
administration primarily for ease of administration
compliance. However, the systemic availability of hydrophilic and or
macromolecular drugs. particularly peptides, administered enterally or
perorally is often too low to have any therapeutic affect Limiting factors
which can decrease overall peroral bioavailability ofa drug include low
solubility or chemical instability in the GIT, high gastrointestinal and or
hepatic metabolism and poor intestinal membrane permeability. Low
peroral bioavailability ofa drug is typically undesirable and can lead to
significant intra— and inter patient variability in drug bioavailability and
therefore therapeutic performance
most
and patient
The mammalian small intestine is composed of“finge‘rlil
and crypts that are covered by a continuous layer of polarized. columnar
epithelial cells. This epithelium, consisting ofa heterogeneous group of
cells, forms the interface between the external environment (the
intestinal lumen) and the interstitial space. The most common epithelial
cell is the enterocyte or the absorptive cell. This cell type is responsible
for the majority of the absorption of both nutrients and drugs which
occurs in the small intestine It is highly polarized with distinct apical
IE980809
and basolateral membranes which are separated by tightjunctions. The
actin—rich tight junctions in conjunction with looser desmosomes
maintain the continuity of the epithelium. Efficient regulation of the
opening and closure of the tight junctions is a key event in the control of
macromolecular movement across the epithelium.
Enterocytes have active transport mechanisms that drive the
absorption of nutrients, electrolytes and water (occurs predominantly
over the surface ofthe villus) and the secretion of electrolytes and water
(occurs mainly in the crypts). The apical membrane has uniform
microvilli measuring approximately 1 pm in height in which
disaccharides and peptidases reside. This membrane also expresses
receptor mediated transport systems (e.g., cobalamin) together with ion,
monosaccharide, amino acid, peptide and fatty acid transporters. The
basolateral membrane, in contrast, has smooth contours with no sugar
and peptide hydrolayses. The Na*/K+ ATPase pump is localized in the
basolateral membrane and permits the vectorial movement of ions and
solutes.
The epithelial cells lining the lumenal side of the GIT are a major
barrier to drug delivery following oral administration However, there are
four recognized transport pathways which can be exploited to facilitate
drug delivery and transport: the transcellular, paracellular, carrier-
mediated and transcytotic transport pathways. The ability ofa drug, such
as a conventional drug, a peptide, a protein, a macromolecule or a nano-
or microparticulate system, to "interact" with one or more of these
transport pathways may result in increased delivery of that drug from the
GlTto the underlying circulation.
Certain drugs utilize transport systems for nutrients which are
the apical cell membranes (carrier mediated route).
may also be transported across the cells in
However, many drugs are
located in
Macromolecules
endocytosed vesicles (transcytosis route).
transported across the intestinal epithelium by passive diffusion either
through cells (transcellular route) or between cells (paracellular). Most
orally administered drugs are absorbed by passive transport. Drugs
which are lipophilic permeate the epithelium by the transcellular route
IE980809
whereas drugs that are hydrophilic are restricted to the paracellular
route.
Paracellular pathways occupy less than 0.1% of the total surface
area of the intestinal epithelium. Further, tight junctions, which form a
continuous belt around the apical part of the cells, restrict permeation
between the cells by creating a seal between adjacent cells. Thus, oral
absorption of hydrophilic drugs such as peptides can be severely
restricted. Other barriers to absorption of drugs may include hydrolyzing
enzymes in the lumen brush border or in the intestinal epithelial cells,
the existence of the aqueous boundary layer on the surface of the
epithelial membrane which may provide an additional diffusion barrier,
the mucus layer associated with the aqueous boundary layer and the
acid microclimate which creates a proton gradient across the apical
membrane. Therefore, new strategies for delivering drugs across the
GIT cell layers are needed, particularly for hydrophilic drugs including
peptides and proteins, and macromolecular drugs.
Numerous potential absorption enhancers have been identified.
For instance, medium chain glycerides have demonstrated the ability to
enhance the absorption of hydrophilic drugs across the intestinal
mucosa (Pharm. Res. Vol 1131148-54 (1994)). However, the importance
of chain length and/or composition is unclear and therefore their
mechanism of action remains largely unknown. Sodium caprate has
been reported to enhance intestinal and colonic drug absorption by the
paracellular route (Pharm. Res. 10:857-864 (1993); Pharm. Res. 5:341-
346 (1988)). U.S. Pat. No. 4,545,161 discloses a process for increasing
the enteral absorbability of heparin and heparinoids by adding non-ionic
surfactants such as those that can be prepared by reacting_ethylene
oxide with a fatty acid, a fatty alcohol, an alkylphenol or a sorbitan or
glycerol fatty acid ester. U.S. Pat. No. 5,229,130 discloses a
composition which increases the permeability of skin to a transdermally
administered pharmacologically active agent formulated with one or
more vegetable oils as skin permeation enhancers.
Often, however, the enhancement of drug absorption correlates
with damage to the intestinal wall. Consequently, limitations to the
IE980809
widespread use of GIT enhancers is frequently determined by their
potential toxicities and side effects. Additionally and especially with
respect to peptide, protein or macromolecular drugs, the "interaction" of
the GITenhancer with one ofthe transport pathways should be transient
or reversible, such as a transient interaction with or opening of tight
junctions so as to enhance transport via the paracellular route.
The invention provides a composition for enhancing the
absorption of a drug from the gastrointestinal tract of an animal
comprising a therapeutically effective amount of a non—ionic vegetable oil
GIT absorption enhancer.
The present invention satisfies the above needs by providing a
composition having a non—ionic vegetable oil GIT absorption enhancer
for increasing the enteral absorbability of drugs, especially oral
absorbability of hydrophilic and macromolecular drugs. The non—ionic
vegetable oil GIT absorption enhancer is capable of enhancing the
uptake of a drug from the gastrointestinal tract so as to allow
therapeutically effective amounts of the drug to be transported across the
GIT of an animal such as a human without significant toxic side effects
The non—ionic vegetable oil GIT absorption enhancer of this
invention is capable of transiently interacting with at least one transport
pathway, perferably the paracellular or transcellular pathway so as to
increase the transport of a drug between or through cells in the GIT. In
one embodiment of this invention, the non—ionic vegetable oil GIT
absorption enhancer of this invention is capable of opening tight
junctions in the GIT, thereby increasing the transport of a drug from the
GIT of an animal into the systemic system via the paracellular pathway
In another embodiment of this invention, the non—ionic vegetable oil GIT
absorption enhancer of this invention is capable of increasing the
transport of a drug via the transcellular pathway.
Preferred non—ionic vegetable oil GIT absorption enhancers
according to this invention are natural vegetable oils or derivatives of the
oils, especially ethoxylated natural vegetable oils in which a polyglycol
chain has been inserted into the triglyceride molecule. Most preferred
IE980809
oils, especially ethoxylated natural vegetable oils in which a polyglycol
chain has been inserted into the triglyceride molecule. Most preferred
enhancers according to this invention are babassu oil, almond oil, maize
oil, palm kernel oil, their ethoxylated derivatives or combinations thereof,
especially the babassu oil, such as the Crovol oils obtained from Croda
Oleochemicals.
Preferred drugs include drugs that, absent the non-ionic
vegetable oil GIT absorption enhancer of this invention, are poorly
absorbed via enteral, especially oral, routes including hydrophilic drugs
or macromolecular drugs such as peptides, proteins or hormones.
Heparin and heparinoids including low molecular weight heparin and
thyrotropin releasing hormone are especially preferred drugs.
The composition according to the invention can be used in a
method of enhancing the bioavailability of a drug following enteral,
preferably oral, administration of the drug comprising enterally
administering a pharmaceutically non-toxic, enhancing amount of a non-
ionic vegetable oil GIT absorption enhancer to an animal either
simultaeously with or prior to the administration of the drug.
The composition according to this invention can be administered
enterally in a conventional solid or liquid pharmaceutical forms, e g ,
tablets, film tablets, capsules, powders, granules, coated tablets or
solutions These can be prepared in a conventional manner and to do
so the drug can be mixed with conventional pharmaceutical auxiliaries.
such as tablet binders, fillers, preservatives, tablet disintegrators, flow
regulators, plasticizers, wetting agnets. disperssants, emulsifiers.
solvents and/or retarding agents.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows the effect of 8 mM Babassu oil added apically at time
zero on TEER of Caco-2 monolayers (cells: day 21, P30-31) over a two
hour period (transport study of [3H]-mannitol). [3H]-mannitol TEER (open
circles; N=9); [3H]—mannitol and 8 mM Babassu oil (closed circles; N=5)
IE980809
Fig. 2 shows the effect of 1 (lg/ml Cytochalasin D added apically
and basolaterally at time zero on TEER of Caco-2 monolayers (control
cells: day 21, P31; cells 8. Cytochalasin D: day 27, P34) over a two hour
period (transport study of [EH]-mannitol). [3H]-mannitol TEER (open
circles; N=9); [3H]-mannitol and 1 pg/ml Cytochalasin D (closed circles;
N=6) (*P=0 04, **P=0.004 and ***P=0.001 (obtained using 2—tailed
unpaired student t—test));
Fig. 3 shows the effect of 8 mM Babassu oil added apically at time
zero on TEER of Caco-2 monolayers (control cells: day 27, P31, cell & 8
mM Babassu oil: day 27, P34) over a two hour period (transport sudy of
[3H]—TRH). [3H]—TRH TEER (open circles; N=4); ); [3H]-TRH and 8 mM
Babassu oil (closed circles; N=5) (*P=0.002, ** P=0.009 and ***
P=0.0001 (obtained using 2—tailed unpaired student t—test));
Fig. 4 shows the effect of 1 pg/ml Cytochalasin D added apically
and basolaterally at time zero on TEER of Caco-2 monolayers (control
cells: day 27, P31; cells 8i Cytochalasin D: day 25, P42) over a two hour
period (transport study of [3H]-TRH). [3H]-TRH TEER (open circles; N=4);
[3H]—TRH and 1 pg/ml Cytochalasin D (closed circles; N=6) ((*P=0.04,
**P=0.0083 and ***P=0.005 (obtained using 2—tailed unpaired student t-
test));
Fig. 5 shows the effect of excess cold TRH added appically at time
zero on TEER of Caco-2 monolayers (control cells: day 27, P31; cells &
excess cold TRH: day 25, P42) over a two hour period (transport study of
[3H]—TRH) [BH]-TRH TEER (open circles; N=4); [‘°’H]-TRH and excess cold
TRH (closed circles; N=6);
Fig. 6 shows the effect of 2 mM Almond oil (A70; —L ), 2 mM
Palm Kernel oil (PK70; —A—), 2 mM Maize oil (M70; —V~) and 2 mM
Babassu oil (BA70G; —O—) added appically at time zero on TEER of
Caco-2 monolayers over a two hour period (transport study of [3H]-TRH)
expressed as % resistance remaining (*statistically significant
difference between Babassu oil TEER results and the remaining oils at
each 30 minute interval except at t=3O min there was no significant
difference in TEER between Babassu oil and Almond oil); and
IE980809
Fig. 7 shows a graph of % of control v. concentration (mM) curves
for the MTT Assay for Almond oil (A70: —¢— ), Palm Kernel oil (PK70: —
l—), Maize oil (M70: —A—), and Babassu oil (BA70G: —o—).
As used in this specification and appended claims, the singular
forms “a", “an” and "the" include plural referents unless the content
clearly dictates otherwise. Thus, for example, reference to "'a vegetable
oil” includes a mixture of two or more vegetable oils, reference to a "a
drug” includes reference to one or more drugs, and the like
As used herein, the term “non—ionic vegetable oil GIT absorption
enhancer" refers principally to a natural vegetable oil or a derivative of the
oil, particularly an ethoxylated vegetable oil in which a poiyglycol chain
has been inserted into the triglyceride molecule, which is capable of
enhancing the transport of a drug, particularly a hydrophilic and/or
macromolecular drug such as a peptide, across the GIT in an animal
such as a human. Such absorption enhancers include but are not
limited to babassu oil, almond oil, maize oil, palm kernel oil, castor oil,
coconut oil, cotton seed oil, jojoba oil, linseed oil, mustard oil, olive oil,
peanut oil, safflower oil sesame oil, soybean oil, sunflower—seecl oil and
wheat germ oil, their ethoxylated derivatives or combinations thereof. As
indicated above preferred oils are babassu oil, almond oil, maize oil and
palm kernel oil, most preferably babassu oil, such as the Crovol oils
obtained from Croda Oleochemicals, England
As used herein, the term "drug" includes any drug appropriate for
administration via the enteral, especially oral, route including
conventional drugs. The term “drug“ also explicitly includes those
entities that are poorly absorbed via enteral, especially oral, routes
including hydrophilic drugs or macromolecular drugs such as peptides,
proteins or hormones including, but not limited to, insulin, calcitonin,
calcitonin gene regulating protein, atrial natriuretic protein, colony
stimulating factor, betaseron, erythropoietin (EPO), interferons such as or
,[30r'y interferon, somatropin, somatotropin, somatostatin, insulin—like
growth factor (somatomedins), luteinizing hormone releasing hormone
(LHRH), tissue piasminogen activator (TPA), thyrotropin releasing
hormone (TRH), growth hormone releasing hormone (GHRH), oxytocin,
IE980809
estradiol, growth hormones, Ieuprolide acetate, factor VIII, interleukins
such as interleukin—2, and analogues thereof and anti—coagulant agents
such as heparin, heparinoids, hirudin, and analogues thereof. The term
”drug" also includes nano— or microparticuiate drug delivery systems in
which a drug is entrapped, encapsulated by, associated with, or
attached to a nano— or microparticle.
As used herein, a "therapeutically effective amount" of a non—ionic
vegetable oil GIT absorption enhancer refers to an amount of enhancer
that allows for uptake of therapeutically effective amounts of the drug via
enterai administration.
EXAMPLE 1
Solubility, osmolarity and pH of various oils. The following oils
were obtained from Croda Oleochemicals, England: Babassu oil
(Crovoi BATOG), Almond oii (Crovol A40; Crovoi A70), Maize oii (Crovoi
lvi4'0; Crovoi M70) and Palm kernel oil (Crovoi PK40; Crovoi PK70).
These oils, which are stable and nonionic in nature, consist of several
vegetable types, some haivng two levels of ethoxylation A polyglycol
chain has been inserted into the triglyceride molecule to impart water
dispersibility or solubility. The solubility of the various oils was
assessed in Hanks + Hepes at 37°C as shown in Table 1.
Table 1
Almond Palm Maize Almond Palm Maize Babassu
Concentration Oil Kermel Oil Oil Kernel Oil Oil
PK4O _ M40 A70 . _ PK7O 1/l7D NEJOG
01 mM soluble insoluble insoluble soluble soluble soluble soluble
mi‘v'i insoluble insoluble insoiubie soluble soluble soluble soluble
1.“. mfv‘. .n§.oi.ibie insoluble insoluble soluble soluble soluble soluble
40 'nlV| I soluble
IE980809
The solublility of the various oils in a 75:25 water to ethanol solution as
assessed as shown in Table 2.
Table 2 ‘
Concentration Almond Oil A40 l Palm Kermel PK4O l Maize Oil M40
— ~ l
mM insoluble l insoluble Insoluble ‘
The osmolarity and pH of a range of concentrations of Babassu
oil in Hank's buffer was examined (0.8 mM — 40 mM). The osmolarities
were in the range of 277-302 mOsm/L (acceptable osmolarity values for
a physiological buffer should be in the range of 280-320 mOsm/L) and
the pH values ranged from pH 7.23 to 7.3 2 mM Almond oil (A70), 2 mM
Maize oil (M70) and 2 mM Palm Kernel oil (PK70) has osmolarity values
of 319, 319 and 322 mOsm/L, respectively, while their respective pH
values were pH 7.33, 7.37 and 7.39
Example 2
Fiux of Kraeber LMW Heparin across Caco—2 monolayers treated
with Babassu of! (BA 70G). The availability of intestinal cell lines which
can be grown as monolayers on permeable filters to an epithelium-
strucure has made it possible to study properties and responses of
epithelial cells without interference of the lamina propria. The Caco-2
cell line is derived from a lung metastasis of a human colon
adenocarcinoma in a 72-year old male (ATCC designation: CCL 248).
When cultured on polycarbonate filters, Caco—2 cells form a confluent
monolayer with several properties characteristic of differentiated
intestinal epithelial cells. The cell layers are morphologically distinct
and have well defined brush borders at their apical surface. They exhibit
characteristic active transport systems and display low permeability to
non—specific transepithelial passage of macromoleucles. Additionally,
the reproducibility and long term viability of the in vitro model, together
with the ability to conveniently perform qualitative and quantitative
IE980809
transport studies across an intact epithelial layer, confer significant
advantages over other in vitro models used for absorption studies. As
such, transport studies of entities such as drugs or absorption
enhancers across Caco—2 monolayers are predictive of transport of
these entities across gastrointestinal tract cell layers in an animal such
as a human.
Caco—2 cells were cultured in Dulbecco’s Modified Eagles
Medium (DMEM) 4.5 g/I glucose supplemented with 1% (v/v) non~
essential amino acids, 10% foetal calf serum (FCS) and 1%
penicillin/streptomycin. The cells were cultured at 37°C and 5% CO2 in
95% relative humidity. The cells were grown and expanded in normal
tissue culture flasks and were passaged once they attained 100%
confluence. The Caco—2 cells were then seeded on polycarbonate filters
(Costar, 12 mm diameter, 0.4 pm pore size) at a density of 5 x 105
cells/cmz and incubated in six—well culture plates with a medium change
every second day for the first six days and subsequently everyday.
Confluent monolayers between day 20 and day 30 post seeding on
filters were routinely used for the transepithelial transport studies.
Part a: Caco—2 monolayers (passage 20-40; 20-30 days post
seeding on filters) were treated apically for 20-30 min with the 8mM and
40mM Babassu oil in Hanks + 25 mM Hepes. The oil was removed and
replaced with Hanks containing 100 IU/ml Kraeber low molecular weight
heparin Samples were taken from the apical side at T=0 and T=120
min and from the basolateral side at T=60 min and T=120 min and
tested in the Factor Xa assay as shown in Table 3. Transepithelial
electrical resistance (TEER) measurements were taken at regular
intervals for 20 min and subsequently every 30 min after the oil was
removed with the Evohmeter (WPI) and chopsticks as shown in Table 4.
The results are expressed as mean 1 S.E. (statistical differences
examined using the two sided independent t—test). The TEER values of
treated monolayers were only significantly different after 20 min,
P<0.0001.
Table 3: Percentage Kraeber low molecular weight heparin transported to the
basolateral side of Caco-2 monolayers aftertreatment with Babassu oil
Time(min) 8mM Babassu oi|,100lU/ml 40 mM Babassu oil. 100 IU/ml
Kraeber hepafltfl\l=3) Kraeberfleparin (_l\l_=:3) (
o-so oi.2§o‘ o.§oo 1 0.160
0-120 046310.061 151010.310
‘heparin found in the basolateral sample from 1 monolayer only
Table 4: Percentage resistance remaining of TEER at time = 0 over 2 hour flux
Control 8 mM Babassu oil, 40 mM Babassu oil,
Time (min) 100 lUlml Kraeber 100 IU/ml Kraeber 100 |Ulm| Kraeber
heparin (N=2) _hepari_(_N=3) heparin (N=3)
0 100 0 1 00.0 100.0 1 00.00 100.0 1 00.00
1 100.0 1 00.0 100 0 1 00.00 A 100 O 1 00.00
3 99.5 1 00.71 99.8 1 00.29 _ 99.3 1 1.30
100.0 1 00.00 80.8 117.60 99.0 11.80
99.01141 80816.70 86811330
929110.00 47711160 i 46716.20
E _. . i5£®_ .. 1%. 1.
_ §0 ¥Z ]0 T 49.7 1 9_23 i } 377112.10 __
90 A 7 74.3 136 30 _ 47.0 1 5 68 39.0 11440 i
120 , §§~§ 1 34.20 39.7 1 9.73 361 .+. 7.12
Part b.‘ 100 lU/ml Kraeber LMW heparin was added apically to
Caco-2 monolayers in the presence of 0.8 mM, 4 mM, 8 mM and 40 mM
Babassu oil at 37°C in an incubator. Samples were taken from the
apical sides at T=0 and 2 hours. Samples were taken from the
basolateral sides at 30 min intervals over the 2 hour period as shown in
Table 5 (0.8 mM babassu oil had no effect on the flux of heparin). TEER
measurements were taken at T=3 min and T210 min and subsequently
every 30 min as shown in Table 6.
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Table 5: Percentage Kraeber low molecular weight heparin transported to the
basolateral side of Caco—2 monolayers in the presence of Babassu oil
Time(min) 4mM Babassu oil. 8 mM Babassu oil, 40 mM Babassu oil,
100 lU/ml Kraeber 100 IU/ml Kraeber 100 IU/ml Kraeber
‘heparin (_N=6) heparin (N=10) hepagn (_N=3l_
o-so 0.000: 0.000 _ o.:L:0.0g4' 037310002 1
sfo-igo 0.678i0.05O 1 1.344:0@ 221910.160
*heparin found in the basolateral samples from 6 out of 10 monolayers
Table 6: Percentage resistance remaining of TEER at time = 0 over 2 hour flux
Control 4 mM Babassu oil. 8 mM Eabassu oil, #40 mM Babassu Oil,
Time 100 IU/ml 100 lUlml Kraeber 100 lU/ml Kraeber 100 lU/ml Kraeber
(min) Kraeber heparin heparin (N=6) heparin (N=10) heparin (N=3)
(N=4l
0 100.0 1 00.0 100 0 -.t 00 00 100.0 :1: O0 00 1000 :t 00.00
3 99.5 i 0.00 1000 :_q.00 94 6 1 3.53 86.5 1 7 73 A
u 99 0 d: 1.40 100 O‘; 0.0_0 100.0 : 0 00 91 5 i 9.33
sgsieoo V 985} 1_9§ 519_:1165 435x915
60 g 9291100 __7si i719 3638:1018 sisiiztg
90 736:21.0 534«_~1@ 3461820 (;7_g_:i175
12p 697:260m 39'/':27O 30919.9 188t10.08
Transmission electron microscopy, TEM, was carried out on the
monolayers which had been treated with O 8 mM, 4 mM, 8 mM and 40 m
Babassu oil for 2 hours. No effect was seen on the integrity of the
monolayers at 0.8 mM or 4 mM Babassu oil. At 8 mM oil, a low
percentage of the junctions were slightly dilated but there was no cellular
damage. Although the resistance measurements had decreased
significantly in the monolayers treated with 40 mM oil, it was not until the
TEM was carried out that is was apparent how toxic this concentration
was to the cells. During preparation for TEM, most of the cells had come
off the filters and those remaining suffered degradation.
The above clearly shows that 8 mM Babassu oil enhances the flux
of heparin across Caco-2 monolayers without having an adverse effect
on the monolayers. There were no differences seen regarding the effect
of the Babassu oil on cell integrity or enhancement of flux using the oil
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as a pretreatment or as a component throughout the two hour flux.
Babassu oil causes a drop in TEER; however, after the oil is removed
and replaced with Hanks, the TEER remained at 50% and it did not
recover. When present throughout the 2 hour flux, it causes TEER to
drop initially followed by a gradual fall. From these results, one could
assume that the enhancement route for heparin is probably
predominantly transcellular as flux appears unrelated to the TEER and
also because heparin is more likely to go across cells because of its
physico-chemical properties.
Example 3
Flux of Kraeber LMW Heparin across rat colon tissue treated with
Babassu or! (BA 70G). Rats of approximately 350g in weight were
dissected and samples of colon tissue were removed and washed in 1 x
Hank's Balanced Salt Bur’-fer (HBSS; Gibco BRL, Cat # 14065-031) . The
tubular segment was cut along the mesenteric border to give a flat
square piece of tissue. The smooth muscle layer was then removed by
blunt dissection to leave an approximate 2.5cm2 patch of epithelium.
The isolated rat colonic mucosae were mounted in Side-by-side
Sweetana—Grass (SG) diffusion chambers in Hanks + NaHCO3. After the
tissue had equilibrated, 100 lU/ml Kraeber LMW heparin (8456 MW) was
added to the apical side of the tissue in the presence of 8 mM Babassu
oil Samples were taken from the apical side at T=O and 120 min
Samples were taken from the basolateral sides at 30 min intervals
(results shown in Table 7) TEER measurements were taken every 30
min and the samples were tested in the Factor Xa assay (results shown
in Table 8).
Table 7: Percentage Kraeber low molecular weight heparin transported to the
basolateral side of rat colon tissue in the presence of Babassu oil
Timetminj 8mM Babassu oil. 100 IU/ml Kraeberheoarin (N=4)
0-60 0.000 t 0.000
60-120 0.195:l;0.077
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Table 8: Percentage resistance remaining of TEER at time = 0 over 2 hour flux
Control (100 IU/ml Kraeber ‘FmM Babassu oil, 100 IU/ml Kraeber
Time (min) heparin (N3)) heparin (N26)
_ 9 V 100. Ii: 00 0 [ 100. : 00.00
_ 96:346 91511025
69 74:t179 _ 85§5i10.15
9_L ‘T 69iti57 880t16.97
120 I 641158 83.511124
As given in Example 2 above, an increase in the flux of Kraeber
LMW heparin across Caco—2 monolayers was detectable at 90 min
whereas there seemed to be a longer lag period with rat colon in that
heparin was only detectable on the basolateral side at 2 hours.
Babassu oil did not have the same effect on the TEER measurements of
the rat colon tissue as it did on the Caco—2 monolayers. When the flux of
Kraeber heparin across Caco—2 monolayers was examined without the
use of Babassu oil, no heparin was detectable in the basolateral
samples.
Example 4
Flux of mannitol across Caco—2 monolayers treated with Babassu
oil {BA70G) or Cytochalasin D. The effect of Babassu oil on the flux of
mannitol (mannitol, D -11 -3H (n)]; Specific activity: 15-30 Ci/mmol; NEN
DuPont) across Caco—2 monolayers was examined by applying 1 uCi/ml
(a concentration of 0.04 nM) mannitol in the presence of 1% Babassu oil
(8 mM) to Caco—2 monolayers prepared as given in Example 2 for 2
hours. The cell monolayers were incubated with prewarmed Hanks
balanced salt solution (HBSS modified by adding 1g glucose/l —_final
concentration 11 mM glucose and 24 mM Hepes, pH 7.4) at 37°C for 30
minutes, adding 1 ml to apical and 2 ml to the basolateral side. At T=O,
apical HBSS was replaced with 1 ml HBSS containing the mannitol
transport solution and the plates were placed in a 37°C incubator on an
orbital shaker. Samples were taken from the apical side at T=0 and
T=120 min and from the basolateral side every 30 min. The resulting
samples were measured by liquid scintillation spectrophotometry using
a Wallac 1409. The apparent permeability coefficient (Paw, cm/sec)
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values were found to increase from 0.79 x 105 for the control (N=6;
mannitol flux across Caco—2) to 2.19 x 106 for mannitol flux across Caco-
2 monolayers in the presence of 1% Babassu oil (N=5) (P =0.001).
TEER measurements (Millicell ERS) were taken throughout the 2 hour
flux at 30 min intervals as shown in Fig. 1. These results demonstrate
that mannitol was without effect on TEER with respect to toss of TEER in
the controls (not treated with Babassu oil). However, a statistically
significant decrease in TEER was induced by treatment with 8 mM
Babassu oil. In the first 60 minutes there was a dramatic reduction of
60% which then became more gradual during the remaining 60
minutes.
Mannitol has been shown to be restricted to transport via
extracellular routes and has proved to be of considerable use in the
quantification of paracellular pathway modification in intestinal model
cell lines. The above resuls confirm that Babassu oil also has a definite
effect on the paracellular pathway as there is a 3-fold increase in the flux
of mannitol in monolayers treated with the oil as opposed to no
treatment with the oil.
An experiment similar to the above transepithelial transport of
mannitol experiment was conducted except that cytochalasin D (1
pg/ml), a potent tight junction opener, was added apically and
basolaterally at time zero instead of the Babassu oil As above. TEER
measurements were taken as shown in Fig. 2. Similar to the Babassu
oil experiment, these results demonstrate that mannitol was without
effect on TEER with respect to loss of TEER in untreated preparations
(not treated with cytochalasin D) A reduction in TEER of 60% was
observed in the presence of the tight junction opener cytochalas_in D.
However, the observed decrease in TEER in the presence of
cytochalasin D was not as significant as that observed when Babassu
oil was added apically at T=0. The apparent permeability coefficient (Paw,
cm/sec) for mannitol increased to 3.15 x 106 (P<0.0001) in the presence
of cytochalasin D. These results show that a similar enhancement of
mannitol flux occurs in the presence of 8 mM babassu oil as was
observed in the presence of Cytochalasin D, indicating a possible
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mechanism of action for babassu oil similar to the tight junction opener,
cytochalasin D.
Example 5
Recovery of Caco—2 monolayers after exposure to Babassu oil
(BA 70G). The ability of Caco—2 monolayers to recover after exposure to
Babassu oil was investigated by treating monolayers with 8 mM
Babassu oil for an incubation period of 2 hours at 37°C in Hanks +
Hepes (similar to Examples 2 and 4 above). After this incubation period,
the monolayers were rinsed gently in complete culture medium and
were replaced in the CO2 incubator at 37°C. TEER measurements were
taken at t, 2, 20 and 24 hours in culture. Untreated monolayers were set
up in Hanks as a control measure (N=3). The resistance
measurements of all the treated monolayers returned to those obtained
originally in culture medium and actually increased with respect to the
control monolayers. Thus, although there was up to a 50% reduction in
TEER across the monolayers after treatment with 8 mM Babassu oil, this
effect was transient and the monolayers were able to recover.
Example 6
Flux of [3H]—TRH across Caco—2 monolayers treated with Babassu
oil (BA 70G) or Cytocha/asin D Thyrotropin releasing hormone (TRH) is
the hypothalamic peptide that regulates the synthesis and secretion of
thyroxine T4 and triiodothyronine from the thyroid gland. TRH absorption
occurs principally in the upper small intestine with little or no absorption
occurring in the middle or lower small intestine. TRH is thought to be
absorbed predominantly passively through a paracellular route between
the cells.
TRH is given currently by intravenous injection in the diagnosis of
disorders of the hypothalmic—pituitary—thyroid axis for mild
hyperthyroidism, hypothyroidism and opthalmic Graves’ disease. It is
also under inventigation in the prevention of neonatal respiratory
distress syndrome.
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The effect of Babassu oil on the flux of TRH (specific activity: 70-90
Ci/mmol; NEN DuPont) across Caco-2 monolayers was examined by
applying 0.12 nM TRH in the presence of 1% Babassu oil (8 mM) to
Caco-2 monolayers prepared as given in Example 2 for 2 hours. The
cell monolayers were incubated as given in Example 4 above. At T=0,
apical HBSS was replaced with 1 ml HBSS containing the TRH transport
solution and the plates were placed in a 37°C incubator on an orbital
shaker. Samples were taken from the apical side at T=0 and T=120 min
and from the basolateral side every 30 min. The resulting samples were
measured by liquid scintillation spectrophotometry using a Wallac 1409.
The apparent permeability coefficient (Paw, cm/sec) values were found to
increase from 0.53 x 106 for the control (N=4; TRH flux across Caco-2) to
2.87 x 105 for TRH flux across Caco-2 monolayers in the presence of 1%
Babassu oil (N=5). TEER measurements (Millicell ERS) were taken
throughout the 2 hour flux at 30 min intervals as shown in Fig. 3. These
results demonstrate that TRH was without effect on TEER with respect to
loss of TEER in the controls (not treated with Babassu oil). However, a
statistically significant decrease of 78.2% in TEER was induced by
treatment with 8 mM Babassu oil during the flux.
An experiment similar to the above transepithelial transport of
TRH experiment was conducted except that cytochalasin D (1 pg/ml), a
potent tight junction opener. was added apically and basolaterally at time
zero instead of the Babassu oil As above, TEER measurements were
taken as shown in Fig 4. Similar to the Babassu oil experiment, these
results demonstrate that TRH was without effect on TEER with respect to
loss of TEER in untreated preparations (not treated with cytochalasin D)
Similar to the mannitol results given in Example 4 above, there was an
initial reduction of 40% in TEER during the first hour of the flux with an
additional 22 % reduction in the remaining hour in the presence of
cytochalasin D. The observed decrease in TEER in the presence of
cytochalasin D was not as significant as that observed when Babassu
oil was added apically at T=0. The apparent permeability coefficient (Paw,
cm/sec) for TRH increased to 5.7 x 106 cm/secin the presence of
cytochalasin D. These results show that a similar enhancement of TRH
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flux occurs in the presence of 8 mM babassu oil as was observed in the
presence of Cytochalasin D.
A further 2 hour transepithelial transport study was carried out as
above for [3H]-TRH (0.12 nM) except that excess TRH (cold, 4.4 mM;
Bachem, UK) was also administered apically. Fig. 5 shows that there is
a similar reduction in TEER of approximately 20% in the presence and
absence of an excess of cold TRH. Statistical analysis carried out at
each time point did not yield any significant difference between TEER
reduction of TRH control cells and that of cells exposed to an excess of
cold TRH. A [3H]—TRH Paw of 0.69 x 106 cm/sec was observed in the
presence of excess cold TRH. No significant difference between TRH
Paw values at low concentrations (0.05-0.1 nM) and high TRH
concentrations (4.4 mM) was observed providing evidence of passive
TRH absorption across the monolayer.
Both [3H]-mannitol and [3H]-TRH control TEER values did not
decrease significantly during the 2 hour flux experiments of Examples 4
and 6 with an overall decrease of 12.8% in resistance of mannitol control
cells and a decrease of 29.2% in TRH control cells Upon addition 1%
(8 mM) babassu oil added apically at time zero, a statistically significant
decrease in the TEER of the Caco—2 monolayers was recorded. Those
cells exposed to the [3H]—mannito| solution containing 8 mM babassu oil
[3H]—exhibited a decrease of 79 4% in TEER. Similarly, those exposed to
[3H]—TRH solution containing 8 mM babassu oil exhibited a reduction of
77.1%.
The results of these studies demonstrate that the flux of TRH, like
mannitol, was linear and did not differ significantly from that of mannitol.
The similarity between mannitol and TRH transport across the Caco—2
monolayers and the similarity of the enhancement of the flux of both
drugs across the monolayers in the presence of Babassu oil evidences
a possible paracellular mechanism of transport for TRH across Caco—2
monolayers.
The results of the cytochalasin D flux experiments provide further
evidence for paracellular transport of TRH. Cells exposed to both [3H1-
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mannitol and cytochalsin D (added apically and basolaterally at time
zero) and those exposed to [3H]-TRH and Cytochalasin D (added apically
and basolaterally at time zero) exhibited a decrease in TEER of 59% and
50% respectively during the 2 hour flux experiments. As predicted, the
tight junction opener, Cytochalasin D caused a statistically significant
enhancement of the flux of both [3H]—mannitol and [3H]-TRH across the
monolayers and similarly increased the Pa“, of both [3H]-mannitol and
[3H]—TRH in the presence of the tight junction opener These results
indicate that Babassu oil enhances TRH absorption in a similar manner
to Cytochalasin D, ie. the Babassu oil mediated enhancement of TRH
absorption was as a result of tight junction opening.
The results of flux experiments with [3H]-TRH in the presence of
excess cold TRH added apically at time zero, yielded a Paw for TRH
which was not significantly different from that obtained for [3H]—TRH at
low concentrations. This result indicates that TRH absorption is
concentration independent, is passive, and occurs through a
paracellular route between the cells.
TRH is typically given in doses of 200-500 pg (100 ug/ml) to reach
a therapeutic plasma concentration of 0.04 mg/L. The surface area of
the filter used in this study was 1.13 Cm‘? and the absorbing surface
area of the small intestine is ~ 125m2. By correlating these two figures,
an estimate of the the TRH concentration required to achieve therapeutic
plasma concentration when administered orally can be made it was
observed that O 2% TRH was transported across the Caco-2 monolayer
in 1 hours. Babassu oil provided an enhancement of the flux to We
transported in 1 hour. For instance. rather than having to use 5 mg/L
solutions of TRH to achieve the peroral therapeutic concentrations, only
0.4 mg/L solutions are required if they are administered with an
enhancing amount of Babassu oil.
Example 7
Flux of heparin across Caco-2 monolayers treated with Maize oil
(M70) or Palm Kernel oil {PK70). Following the procedures of the 2 hour
flux experiments of Example 2, Part b, unfractionated heparin (600 lU/ml)
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was added apically to Caco—2 monolayers in the presence of 0.1 mM, 1.0
mM, 20 mM and 3 mM Maize oil and 0.1 mM and 4 mM Palm Kernel oil.
TEER measurements were taken every 30 minutes and the l0ul
aliquots of the apical and basolateral samples were frozen at —20°C until
ready to analyse Heparin transport across the monolayers was
measured using the FXA heparin assay. No transport of heparin across
the monolayers was observed in the presence of Maize or Palm Kernel
Oil at these concentrations.
Example 8
Flux of [SH]-mannitol or [‘3H]—TRH across Caco-2 monolayers
treated with Maize oil (M70), Palm Kernel oil (PK 70), Almond oil (A70) or
Babassu oil (BA 70G). Following the procedures of the 2 hour flux
experiments of Example 4 above, [3H]-mannitol (0.04 nM) was added
apically to Caco—2 monolayers in the presence of 0.1 mM, 5.0 mM, and
mM Almond Oil, 0.1 mM, 2 0 mM. 2.5 mM, 50 mM and 10 mM Palm
Kernel oil as well as 0.1 mM, 1 mM and 2 mM Maize oil TEER
measurements were taken every 30 minutes for each experiment. For
Almond oil and Palm Kernel oil at 5 and 10 mM, approximately a 70%
reduction in TEER was observed which was constant during the course
of the two hour flux. Based on the marked reduction in TEER and evident
toxicity at 5 and 10 mM, a lower concentration of 2 mM was chosen. Flux
of 2 mM Maize oil resulted in a linear decrease in TEER of approximately
% at each 30 minute interval
Fig. 6 shows the effect of apical administration of [3H]—TRl-I (0.2
nM) in the presence of Almond oil, Palm Kernel oil, Maize oil and
Babassu oil (each oil at a concentration of 2 mM) following the flux
procedures given in Example 6 above. Statistically significant .
differences existed between Babassu TEER results and those of the
other oils (2 mM) at each 30 minute interval except between Babassu oil
and Almond oil at the T=30 sampling No significant differences in
TEER was observed between Almond oil, Palm Kernel oil and Maize oil.
The average 30% reduction in TEER observed for Almond, Palm Kernel
and Maize oils at 2 mM during the 2 hour flux was not evident in the
presence of Babassu oil (2 mM).
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Apical-to-basolateral fluxes of the hydrophilic marker molecule 3H-
mannitol and 3H-TRH in the presence of each of the four oils at a
concentration of 2mM demonstrated a 2 to 3 fold increase in transport
across Caco—2 monolayers. A summary of these transepithelial
transport results is given in Table 9.
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Table 9: Summary of Transepithelial Transport Results for Various Oils
W F F Id
No. of Pm, cmlsec % Transport . O .
Drug [Enhancer exp X 105 isEM per hour lncrease ln
' Transport
[3H]-mannitol control (0 04 nM) 4 0 79 i 0.03 0 28
Mannitol & Almond Oil
2 2 _§
[3H]-mannitol Sr Almond Oil (O.1mM) 3 1.970 t 0.68 2 45
0.07
(31-1}-mannltol 3. Almond on (5.0 mM) 3 1 91 :r 0.69 2 46
0.02
[3H]-mannitol & Almond oll (10 mM) 3 7 413 i 2.67 9 56
0.41
Mannitol 8. Palm Kernel Oil ‘_ 2 V
[°H]—mannitol 8. Palm Kernel (0.1 mM) 3 E‘ I 2.83 i 0.52 1.02 3.64
[3H]-mannitol 3. Palm Kernel (2.0 mM) 4 2.28 2 0 82 2 9
0.136
{°H1-mannllol & Palm Kernel (2.5 mall" 4 2.49 : 0.89 3 17
0.143
[3H]-mannltol & Palm Kernel (5.0 mM) 3 5 87 i 0.54 2.11 7 56
[3H]-mannitol & Palm Kernlel (10 mM) 3 13.63 i 4 92 17 55
2.42
Mannitol 8. Maize Oil
[3H]—rl'Iannltol & Maize Oil (O.1mM) 2 3 39 i 0.25 1 22 4 35
[3H]-mannitol 3. Maize Oil (1 0 mM) 2 3 625 1 1 31 4 67
0 19
[3l-ll-mannrtol & Malze Oil (2.0 mM) 2 6 175 2 2 23 7 96
2 73
["'H]-‘FRI-l control (0 2 nM) 2 0.72 i 0.26
0.025
[3H]-TRH & Almond Oil (2.0 mM) 4 1.45 1- 0.08 0.52 2
[3H]—TRH & Maize Oil (2.0 mM) 3 2.203 L 0.79 3 03
0.48
[°H]-TRH 3. Palm Kernel Oil (2 0 mM) 4 1 146 2 0.412 1 58
0.09
[3H]-TRH & Babassu Oil (2 0 mM) 4 1.189 2 0.43 1 65
0.08
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Example 9
Recovery of Caco—2 monolayers after exposure to Palm Kernel Oil
(PK 70), Almond Oil (A70), Maize Oil (M70) and Babassu oil (BA 70G).
The ability of Caco—2 monolayers to recover after exposure to various oils
at differing concentrations was investigated by treating monolayers with
an oil for an incubation period of 2 hours at 37°C in Hanks + Hepes
(similar to Examples 2, 4 and 8 above). The TEER of the Caco—2
monolayers was first recorded in culture and the monolayers were
incubated in prewarmed Hanks for 30 minutes after which the
resistance was recorded. The apical Hanks was removed and replaced
with 1 ml of a particular oil at a particular concentration. Resistance
measurements were recorded at 30 minute intervals throughout the two
hour incubation period. After this incubation period. the monolayers
were rinsed gently in prewarmed complete culture medium and were
replaced in the CO2 incubator at 37°C. TEER measurements were taken
at 1, 20 and 24 hours in culture. Untreated monolayers were set up in
Hanks as a control measure (N=3). Following two hour flux experiments
with the various oils at concentrations ranging from 0.1 to 10 mM, Caco—
2 monolayers once replaced in culture conditions recovered with 24
hours (except for Maize oil at 2 mM). Table 10 summarizes the results of
these recovery experiments
Example 10
Toxicity Tests for Palm Kernel Oil (PK 70), Almond Oil (A70), Maize
Oil (M70) and Babassu oil (BA FOG) Two tests were employed to identify
the toxic potential of the various oils used in the preceding examples.
The Neutral Red Toxicity Test assesses cell viability based on
lysosomal integrity. The neutral red dye is incorporated into the intact
Iysosomes of living cells whereas dead cells no longer possess the
uptake system necessary to carry out this process. The amount of
spectrophotometrically—measured dye uptake is indicative of the
numbers of viable cells
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Table 10: Results of Recovery Experiments
| 0.1 mM 1 2% I 5 mM |_ 10 mM
Control _ g _ _
Tinlg: Hours __ % Resistance Remaining '
A 1 78.3 _ J
2 T 88.9 é . _
21 _ >100 i _ __ _
i i _ _Ni £4 N=3 N=3
Palm Kerelfl
‘nme_ Hours i% Resistance Remaining __ _
_i1 72 3 18 0 ‘I3 0 5 _
i 20 105.8 95 0 129.9 ‘I72
24 g 92.0 101.0 985 _ _ i 123
Almond Oil (A70)
Time_ Hours % Resistance Remaining
1 62.7 70.9 18
88.2 ____ 144.8 232
24 88.8 136.5 184
Maize Oil 1M70L
Time_ Hours “V _ % Resistance Remaining
; 1 84.6 _i 20 28 65 Z 15 65 A
_ _ A3 _ . . l3j._: 2 2 A
24 95.2 52_ A 1E9 10 2 _
Babassu Oil 7[_B(A70G_) _ V 7 ‘
Time. H095 _ % Resistance Remaining
1 . 1 Se _ l
4 l 6% . l
67 lg
24 . 92 i
For the Neutral Red Toxicity Test, confluent cells are harvested
(e.g., using 2 ml trypsin for approximately 4 min at 37°C) and counted
using standard operating procedures such as the trypan blue dye
exclusion test. A 100 pl cell susension containing 105 cells was added
to each well of a 96 well plate; 3 wells containing 100 pl medium only
was used as a blank. The cells were incubated for 24 hours at 37°C.
Appropriate dilutions such as 1:6 dilution of 0.6, 30, 80 mM Almond oil
and Palm Kernel oil was carried out to yield final concentrations (e.g.,
0.1, 5 and 10 mM) and 20 iii of each concentration was added to the 100
pl cell suspension in each well. Following incubation of the cells for two
hours, the tissue culture medium was removed and the cells were
Table 11: Results of Neutral Red Toxicity Test
Conc. (mM) % of Control 7 7 _
i— — —. Alngnd Oil —-:alm Ke;;OilTPK70) Babassu Oil
(A70) (BA70G)
O 1 98 3 94.06
i 0.8 _i i i _i 100
_5 A _21 3% A 2905 _ f
8 T ‘U 100
I 21 77 32 76 l
40 9
IE980809
washed with 100 pl warm PBS. 100 pl of neutral red solution was
added to each well and incubated at 37°C for 4 hours at which point the
medium was removed and the cells were rinsed with neutral red fixative.
Following removal of the fixative, 100 pl of neutral red solubilization
solution was added to each well for 10 minutes and the absorbance
was read on a spectrophotometer at 570 nm. Table 11 provides the
results of the Neutral Red Toxicity Test for various concentrations of
Almond oil, Palm Kernel oil and Babassu oil.
The MTT Assay measures mitochondrial dehydrogenase (MDH)
activity as a marker of cell viability. MDH is only present in viable cells
and can convert the yellow tetrazolium salt MMT to a purple crystalline
formazan product. The amount of this product is directly proportional to
the number of viable cells.
For the MTT Assay, cells were harvested and counted using
standard procedures. 100 pl of cell suspension was added to 48 wells
and the cells were incubated at 37°C for 24 hours Appropriate dilutions
are made of the various oils and 20 pl of each concentration was added
to the 100 pl suspension in each well. Following a two hours incubation
period, 10 pl of the MTT labelling reagent is added to each well for 4
IE980809
hours. 100 pl solubilization solution is added to each well, the plate is
incubated for 24 hours and the absorbance of each well is measured
using a spectrophotmeter at 570 nm. Table 12 and Fig. 7 provide a
summary of the MTT Assay results for various concentrations of Almond
oil, Palm Kernel oil, Maize oil and Babassu oil. The results of the MTT
toxicity test indicate that between 2 and 3 mM there is a significant
increase in the toxic potential of Almond oil, Maize oil and Palm Kernel
oil. However, at concentrations ranging from up to 10 mM, it appears
that Babassu oil is considerably less toxic and is miminally toxic (9%) at
mM. Thus, for Almond oil, Maize oil and Palm Kernel oil, a narrow
concentration range around 2-3 mM is necessary to avoid marked toxicity
while still maintaining a 2-3 fold increase in transport across Caco—2
monolayers demonstrated by all four oils. Babassu oil, however, is
much less toxic at a concentration five fold higher than the toxicity limit for
the other oils. This finding is important because the limits in the use of
any penetration enhancer may be dictated by their potential toxicities.
Table 12: Results of MTT Assay
M _ % of Control
Conc. (mM) Almond oil i Palm Kernel Maize oil Babassu oil
_ l__A]0l _ oil (PK70) (M70) (BATOG)
0.1 87.39 97.33 . 97.96 93 62
1 : 102.26 1 98.19 1 93.98 89.48
2 59.34 72.5 79 71 914
2.5 19.07 49.62 90.29
3 l 23.67 23.8 23.55 88.3
4 9.45 6.74 9.85 90
11.4 3.52 13.13 89.8
6 V _ 7.8 76.84
8.84 0.0472 a 4.83 81.39
Structural analysis was also used to measure the toxicity of the
oils. TEMs were taken of Caco—2 monolayers subsequent to 2 hour
exposure to palm Kernel oil (PK70) and Maize oil (M70), both at a
concentration of 2 mM. The integrity of the monolayers was intact.
However, dispersion of the microvilli and some junction opening was
apparent.
IE980809
The ability of the Babassu, Almond, Maize and Palm Kernel oils to
enhance the flux of the paracellular marker mannitol and TRH implies
that this enhancement may be mediated by opening tight junctions.
0.7% per hour transport of Kraeber LMW heparin across Caco-2
monolayers was demonstrated in the presence of Babassu oil (8 mM).
The most likely path for heparin, due to its molecular weight of 20 Kd, is
transceliular. Therefore the mechanism of action for the non-ionic
vegetable oil GIT absorption enhancers of this invention is unclear
although they appear to be able to enhance the permeation of agents
across or between cells in the gastrointestinal tract according to the
physicochemical properties of the particular drug and with minimal toxic
effects and transient effects on the cell layers in the gastrointestinal tract.
28 IE980809
CLAlMS:-
A composition for enhancing the absorption of a drug from the
gastrointestinal tract of an animal comprising a therapeutically effective
amount of a non-ionic vegetable oil GIT absorption enhancer,
A composition according to Claim 1, wherein the absorption enhancer is a
natural vegetable oil or derivative thereof.
A composition according to Claim 2, wherein the absorption enhancer is
selected from babassu oil, almond oil, maize oil, palm kernel oil,
ethoxylated derivatives thereof and combinations thereof.
A composition according to Claim 3, wherein the absorption enhancer is
babassu oil or a derivative thereof.
A composition according to any preceding claim, wherein the drug is
selected from hydrophilic drugs and macromolecular drugs.
A composition according to Claim 5, wherein the drug is selected from
peptides, proteins and hormones.
A composition according to Claim 5, wherein the drug is heparin or a
heparinoid.
A composition according to Claim 6, wherein the drug is thyrotropin
releasing hormone
A composition according to any preceding claim, wherein the composition
is an oral composition.
A composition according to Claim 1 for enhancing the absorption of a drug
from the gastrointestinal tract of an animal, substantially as hereinbefore
described and exemplified.
ANNE RYAN 8: CO
AGENTS FOR THE APPLICANTS
IE980809
COMPOSITION FOR ENHANCING TRANSPORT ACROSS GASTROINTESTINAL
TRACT CELL LAYERS
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
USUNITEDSTATESOFAMERICA01/10/19976 | |||
US6061897P | 1997-10-01 | 1997-10-01 |
Publications (3)
Publication Number | Publication Date |
---|---|
IE19980809A1 true IE19980809A1 (en) | 2000-10-04 |
IE980809A1 IE980809A1 (en) | 2000-10-04 |
IE83587B1 IE83587B1 (en) | 2004-09-22 |
Family
ID=
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