WO1998002451A1 - Oligopeptide transporters - Google Patents
Oligopeptide transporters Download PDFInfo
- Publication number
- WO1998002451A1 WO1998002451A1 PCT/DK1997/000312 DK9700312W WO9802451A1 WO 1998002451 A1 WO1998002451 A1 WO 1998002451A1 DK 9700312 W DK9700312 W DK 9700312W WO 9802451 A1 WO9802451 A1 WO 9802451A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- ala
- asp
- gly
- cys
- lys
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06104—Dipeptides with the first amino acid being acidic
- C07K5/06113—Asp- or Asn-amino acid
-
- 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
- A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
Definitions
- the present invention relates to di- and tripeptides and their use as pro-moities to facilitate the transmembrane transport of drugs, in particular of small molecular weight drugs and more particular poorly absorbed small molecular weight drugs
- the oligopeptide transporter is expressed in mammalian small intestinal epithelia and a number of biomembranes, and is instrumental in the process of ammo acid absorption via active uptake of digested di- and tripeptides from the gastrointestinal lumen ( 1 , 2)
- This transporter is energized by a Na ⁇ - independent H * -grad ⁇ ent, which is generated within the acid microclimate located adjacent to the apical surface of jejunal microvilh (2, 3)
- the oligopeptide transporter has an immense number of potential di- and tripeptide substrates (4)
- Various types of peptidomimetics have been shown to serve as efficient substrates for the oligopeptide transporter, including ⁇ -lactam antibiotics (5-7) , angiotensm converting enzyme inhibitors (8) , and rennm inhibitors (6) , yet, free ammo acids or intact tetrapeptides are not substrates for this transporter
- oligopeptide transporter displays a preferential affinity for L/L-configured dipeptides as opposed to L/D, D/L, and especially D/D combinations ( 1 0, 14, 1 7) .
- the structural requirements of peptides and peptidomimetics influencing their ability to interact with the oligopeptide transporter have been investigated, e.g. , cychzation ( 1 4), N-termmal ⁇ -am ⁇ no modification (4, 8) and assessment of hydrophobic potential (4, 1 5) ; but, it has not yet been determined if covalent side-chain modification of one of the ammo acids in a linear di- or tripeptide results in a loss of affinity for the oligopeptide transporter.
- este ⁇ fication of the aspartic acid in D-Asp-Ala does not interfere with its ability to inhibit the uptake of [ 14 C]glycylsarcos ⁇ ne ([ 14 C]Gly-Sar) , an enzymatically stable dipeptide having a high affinity for the oligopeptide transporter expressed in Caco-2 monolayers ( 1 6)
- Fetal bovine serum FBS
- D-MEM Dubeicco's Modified Eagle's Medium
- NEAA 100x non-essential ammo acids
- trypsin 0.25%
- -EDTA 1 mM
- penicillin 1 0,000 U/ml
- streptomycin 1 0,000 ⁇ g/ml
- HBSS Hank's Balanced Salt Solution
- MES N-2-hydroxyethylpoperaz ⁇ ne-N'-2-ethanesulfon ⁇ c acid
- BSA Bovine Serum Albumin
- Caco-2 epithelial cells were obtained from the ATCC (Rockville, MD), and were used between passages 21 and 40. They were seeded into tissue culture treated TranswellsTM (4.7 cm 2 , 0.4 / pore size, Costar Corp., Cambridge, MA) at a density of 1 0 5 cells/cm 2 Cells were maintained in a humidified 5% C0 2 in air atmosphere at 37°C, and were cultured in Dubelco's Minimal Essential Medium (D-MEM) supplemented with 1 0% fetal bovine serum (FBS), penicillin/streptomycin ( 1 00 U/ml and 1 00 /vg/ml, respectively), 1 % L-glutamine, and 1 % NEAA.
- D-MEM Dubelco's Minimal Essential Medium
- FBS fetal bovine serum
- penicillin/streptomycin 1 00 U/ml and 1 00 /vg/ml, respectively
- 1 % L-glutamine 1 %
- TEER transmembrane electrical resistance
- Cell monolayers could be maintained at a constant resistance for at least 7 days without suffering leakage between apical and basal compartments (as determined by [ 14 C]Mann ⁇ tol flux) or a loss in TEER
- the total amount of protein on each Transwell filter was calculated using the Lowry method to be 0 42 mg/cm 2
- Boc-D-Asp(OtBu)-OH was liberated from its DCHA salt by standard procedure yielding a colourless oil which was used without further purification
- Boc-D-Asp(OtBu)-Ala-OtBu 1 .23 g Boc-D-Asp(OtBu)-OH (4.25 mmol) was dissolved in 20 ml fresh distilled DMF at room temperature together with 0.574 g HObt (4.25 mmol) and 1 .365 g TBTU (4.25 mmol) The mixture was then stirred at room temperature for 5 mm before adding 0.772 g H-Ala-OtBu (4.25 mmol) and 1 .648 g DIPEA ( 1 2.75 mmol) . The resulting mixture was stirred for 2 hr at room temperature and then evaporated to dryness.
- H-D-Asp-Ala-OH The oil was dissolved in 20 ml DCM and 20 ml concentrated TFA was added and the mixture stirred for 2 hr at room temperature. The solvent was then evaporated under reduced pressure and the resulting oil was extracted with 3 x 1 0 ml ether, which turned the oil into 600 mg crystals, 70% in overall yield. The purity was verified by TLC and HPLC and was found to be better than 98%. The product was identified by ' H-NMR
- H-D-Asp(OcHex)-Ala-OH was prepared using the above described procedure resulting in a 75% overall yield (91 0 mg) . The purity was verified by TLC and HPLC and was found to be better than 98%, and the product was identified by ' H-NMR.
- H-D-Asp(OBzl)-Ala-OH was prepared using the above described procedure resulting in a 59% overall yield (735 mg) . The purity was verified by TLC and HPLC and was found to be better than 98%, and the product was identified by 1 H-NMR.
- the amount of uptake and transport of [ 1 C]Gly-Sar following a 2 hr incubation was 3.01 ⁇ 0.1 1 and 0.98 ⁇ 0.06 pmol/mg protein/mm, respectively.
- the degree of [ 14 C]Gly-Sar uptake and transport was significantly less, i.e. , 1 .72 ⁇ 0.1 0 and 0.51 ⁇ 0.1 3 pmol/mg protein/mm, respectively.
- the values are corrected for [ 1 C]Mann ⁇ tol flux across the monolayers, which was 0.06 ⁇ 0.02 for the pH 6 and 0.1 1 ⁇ 0.02 pmol/mg/min for the pH 7.4 experiments.
- affinities of a variety of dipeptides and ammo acids for the oligopeptide transporter were assessed (Fig. 2)
- the two tested L/L-configured dipeptides, Gly-Sar and Gly-Pro inhibit the apical uptake of [1 4C]Gly-Sar by > 90%.
- D/L- or L/D-configured dipeptides demonstrate a reduced affinity for the oligopeptide transporter relative to the L/L-configured dipeptides, nonetheless, they were all able to inhibit [ 1 C]Gly-Sar uptake by > 75 %
- two L-ammo acids, L-Tyr and L-Phe were tested for their ability to inhibit [ 14 C]Gly-Sar uptake in an identical manner, and as is shown in Fig. 2, they were both unable to inhibit [ 14 C]Gly-Sar uptake to any significant degree.
- D-Asp-Ala, D-Asp(cHex)-Ala, and D-Asp(BZ)-Ala were tested in order to assess their respective abilities to inhibit the apical uptake of [ 14 C]Gly-Sar into Caco-2 monolayers during either a 1 5 or a 1 20 mm experiment.
- the extent of inhibition of [ , C]gly-sar uptake is > 95 % for all three compounds following 1 5 mm of competitive inhibition, and > 80% following 1 20 mm of competitive inhibition.
- Equation 1 P (C, - C w )/C w x V w /V 0
- C, and C w represent the concentration of D-Asp(cHex)-Ala and D-Asp(BZ)- Ala in the aqueous buffer phase before and after distribution, respectively;
- V w represents the volume of the aqueous phase;
- V 0 is the volume of the octanol phase
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU34344/97A AU3434497A (en) | 1996-07-16 | 1997-07-16 | Oligopeptide transporters |
EP97930366A EP0923603A1 (en) | 1996-07-16 | 1997-07-16 | Oligopeptide transporters |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DK79096 | 1996-07-16 | ||
DK0790/96 | 1996-07-16 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1998002451A1 true WO1998002451A1 (en) | 1998-01-22 |
Family
ID=8097589
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/DK1997/000312 WO1998002451A1 (en) | 1996-07-16 | 1997-07-16 | Oligopeptide transporters |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP0923603A1 (en) |
AU (1) | AU3434497A (en) |
WO (1) | WO1998002451A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1212619A1 (en) * | 1999-09-14 | 2002-06-12 | Xenoport, Inc. | Substrates and screening methods for transport proteins |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996005863A1 (en) * | 1994-08-19 | 1996-02-29 | La Region Wallonne | Compounds, pharmaceutical composition and diagnostic device comprising same and their use |
-
1997
- 1997-07-16 WO PCT/DK1997/000312 patent/WO1998002451A1/en not_active Application Discontinuation
- 1997-07-16 AU AU34344/97A patent/AU3434497A/en not_active Abandoned
- 1997-07-16 EP EP97930366A patent/EP0923603A1/en not_active Withdrawn
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996005863A1 (en) * | 1994-08-19 | 1996-02-29 | La Region Wallonne | Compounds, pharmaceutical composition and diagnostic device comprising same and their use |
Non-Patent Citations (1)
Title |
---|
DRUG DELIVERY, Volume 1, 1993, PHILIP L. SMITH et al., "Explotation of the Intestinal Oligopeptide Transporter to Enhance Drug Absorption", pages 103-111. * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1212619A1 (en) * | 1999-09-14 | 2002-06-12 | Xenoport, Inc. | Substrates and screening methods for transport proteins |
EP1212619A4 (en) * | 1999-09-14 | 2004-12-08 | Xenoport Inc | Substrates and screening methods for transport proteins |
US7413536B1 (en) | 1999-09-14 | 2008-08-19 | Xenoport, Inc. | Substrates and screening methods for transport proteins |
Also Published As
Publication number | Publication date |
---|---|
AU3434497A (en) | 1998-02-09 |
EP0923603A1 (en) | 1999-06-23 |
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