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1

Electronic Resource

Colonic Absorption and Bioavailability of the Pentapeptide Metkephamid in the Rat (1994)

Langguth, Peter ; Breves, Gerhard ; Stökli, Arthur ; [et al.]

Springer

Pharmaceutical research 11 (1994), S. 1640-1645

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ISSN: 1573-904X

Keywords: intestinal absorption ; colonic delivery ; peptides ; intestinal microflora

Source: Springer Online Journal Archives 1860-2000

Topics: Chemistry and Pharmacology

Notes: Abstract The concept of delivering systemically active peptide drugs to the colon in order to improve their oral absorption requires reasonable peptide permeability of the large intestinal wall and stability against the activity of the colonic microflora. In addition, the role of hepatic extraction needs to be addressed. In this study the absorption of the pentapeptide metkephamid following single pass perfusion of rat ascending colon was investigated by monitoring its disappearance from the large intestine and simultaneous appearance in the portal vein, the hepatic vein and the aorta. In addition its stability against colonic microflora was tested in vitro using pig caecal contents. Metkephamid was absorbed from the large intestine and appeared in the blood circulation; peptide concentrations in the portal vein increased over-proportionally with increasing perfusate concentrations (0.1 − 4.6 mmol/L) from 0.19 µg/mL ± 0.12 (SD, n = 7) to 31.6 µg/mL + 20.65 (SD, n = 4), respectively, and thus suggesting a saturable transport or metabolism. Concentrations in the hepatic vein were significantly lower than in the portal vein, hepatic extraction ratios were 0.35 ± 0.14, 0.61 ± 0.18 and 0.62 ± 0.28 (SD, n = 4) for 0.1, 0.5 and 1.0 mM metkephamid perfusate concentrations, respectively. In the anaerobic colon metabolism model the degradation half-life of the peptide was 14.9 hours, thus, indicating relative stability in the bacterial environment of the colon. The results of the present study encourage further investigations on colonic delivery of peptide drugs.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1023/A:1018974107844

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2

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Overexpression of Human Intestinal Oligopeptide Transporter in Mammalian Cells via Adenoviral Transduction (1998)

Hsu, Cheng-Pang ; Hilfinger, John M. ; Walter, Elke ; [et al.]

Springer

Pharmaceutical research 15 (1998), S. 1376-1381

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ISSN: 1573-904X

Keywords: gene expression ; hPepTl ; Caco-2 cells ; adenovirus ; drug screening

Source: Springer Online Journal Archives 1860-2000

Topics: Chemistry and Pharmacology

Notes: Abstract Purpose. Our goals are to establish an in vitro screening system and to evaluate a new approach in improving oral absorption of peptides and peptide-like drugs by overexpression of the human intestinal oligo-peptide transporter (hPepTl). This study characterizes the expression of hPepTl in human intestinal Caco-2 cells, rat intestinal epithelial cells (IEC-18), and human cervix epithelial cells (Hela) after adenoviral transduction. Methods. A recombinant replication-deficient adenovirus carrying the hPepTl gene was made and used as a vector for the expression of hPepTl. The increase in the uptake permeability of cephalexin and Gly-Sar was determined. The effects of time, dose, apical pH, and substrate specificity were evaluated. Results. A significant increase in the uptake permeability of Gly-Sar and cephalexin was found in all three cell lines after viral transduction. The increase of Gly-Sar permeability in Hela, IEC-18, and Caco-2 cells was 85-, 46-, and 15-fold respectively. Immunoblotting using an antibody against hPepTl detected high levels of a 85-98-kDa protein in all three infected cell lines. Substrate permeability was dependent on time of infection, inward pH gradients, and multiplicity of infection (MOI). Decreased infectivity and lower hPepTl expression were observed in differentiated Caco-2 cells. The uptake was inhibited by dipeptides and β-lactam antibiotics but not amino acids. Conclusions. Adenoviral infected Hela cells displayed a pronounced level of hPepTl expression with a low background and high specificity to dipeptides. These features make this system a useful tool for screening of potential substrates. The success of overexpression of hPepTl in Caco-2 and IEC-18 cells may lead to a novel approach in improving oral absorption of peptides and peptidomimetic drugs.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1023/A:1011993303397

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3

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Inhibition of Succinimide Formation in Aqueous Zn-rHirudin Suspensions1 (1999)

Gietz, Ursula ; Arvinte, Tudor ; Alder, Ruth ; [et al.]

Springer

Pharmaceutical research 16 (1999), S. 1626-1632

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ISSN: 1573-904X

Keywords: recombinant hirudin ; protein formulation ; protein stability ; capillary electrophoresis ; cyclic imide

Source: Springer Online Journal Archives 1860-2000

Topics: Chemistry and Pharmacology

Notes: Abstract Purpose. The formation of succinimide intermediates at Asp-Gly sites and their hydrolysis products, e.g., isoAsp isomers, represents a common source of microheterogeneity in therapeutic proteins. Here we report on the stabilization effect of a zinc chloride induced precipitation of recombinant hirudin HV1 (rHir), an anticoagulant protein. Methods. rHir was precipitated by zinc chloride at neutral pH to form a Zn-rHir suspension. An Arrhenius-type study (at 50, 40, 30, and 25°C) and a 4°C stability study were performed. Monitoring of rHir, rHir succinimides at Asp33-Gly34 (Q5) and Asp53-Gly54 (Q4), and further side products was by capillary electrophoresis (CE). Results. The activation energies of rHir degradation in both aqueous rHir solution and Zn-rHir suspension were similar, i.e. 104.5 and 110.3 kJ/mol, respectively. Zn-rHir suspension demonstrated improved shelf-life stability (t90%, 95% confidence limit) versus rHir solution, i.e., 23 versus 3 days at 25°C and 292 versus 147 days at 4°C, respectively. In rHir solution, Q4 (Asp53-Gly54 succinimide) levels were slightly above Q5 (Asp33-Gly34 succinimide) levels. In Zn-rHir suspension, however, Q4 succinimide levels dropped markedly whereas Q5 levels were not affected. Correspondingly, in Zn-rHir isoAsp53-rHir levels were reduced but not isoAsp33-rHir levels. Conclusions. In Zn-rHir suspensions, interactions of zinc and rHir show site-specific inhibition of succinimide formation only at Asp53-Gly54 (Q4), located in the highly flexible C-terminal tail of rHir. In contrast, succinimide formation at Asp33-Gly34 (Q5), located in a less flexible loop domain is not affected, reflecting steric hindrance.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1023/A:1018925209080

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4

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Proteolysis of Human Calcitonin in Excised Bovine Nasal Mucosa: Elucidation of the Metabolic Pathway by Liquid Secondary lonization Mass Spectrometry (LSIMS) and Matrix Assisted Laser Desorption lonization Mass Spectrometry (MALDI) (1996)

Lang, Steffen R. ; Staudenmann, Werner ; James, Peter ; [et al.]

Springer

Pharmaceutical research 13 (1996), S. 1679-1685

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ISSN: 1573-904X

Keywords: human calcitonin ; salmon calcitonin ; peptide metabolism ; nasal metabolism ; nasal mucosa ; metabolic pathway ; mass spectrometry ; MALDI-MS ; LSIMS ; LC-MS

Source: Springer Online Journal Archives 1860-2000

Topics: Chemistry and Pharmacology

Notes: Abstract Purpose. Two calcitonins, i.e. human calcitonin (hCT) and, for comparison, salmon calcitonin (sCT), were chosen as peptide models to investigate nasal mucosal metabolism. Methods. The susceptibility of hCT and sCT to nasal mucosal enzymes was assessed by in-and-out reflection kinetics experiments in an in vitro model based on the use of freshly excised bovine nasal mucosa, with the mucosal surface of the mucosa facing the peptide solution. The kinetics of CT degradation in the bulk solution was monitored by HPLC. Peptide sequences of the main nasal metabolites of hCT were analyzed by using both liquid secondary ionization mass spectrometry (LSIMS), following HPLC fractionation of the metabolites, and matrix-assisted laser desorption ionization mass (MALDI) spectrometry. For sCT, the molecular weights of two major metabolites were determined by LC-MS with electrospray ionization. Results. Both CTs were readily metabolized by nasal mucosal enzymes. In the concentration range studied metabolic rates were higher with hCT than with sCT. Presence of endopeptidase activities in the nasal mucosa was crucial, cleaving both calcitonins in the central domain of the molecules. Conclusions. Typically, initial metabolic cleavage of hCT in nasal mucosa is due to both chymotryptic- and tryptic-like endopeptidases. The subsequent metabolic break-down follows the sequential pattern of aminopeptidase activity. Tryptic endopeptidase activity is characteristic of nasal sCT cleavage.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1023/A:1016492723930

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5

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Oral Absorption of Peptides: The Effect of Absorption Site and Enzyme Inhibition on the Systemic Availability of Metkephamid (1994)

Langguth, Peter ; Merkle, Hans P. ; Amidon, Gordon L.

Springer

Pharmaceutical research 11 (1994), S. 528-535

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ISSN: 1573-904X

Keywords: absorption ; peptides ; metkephamid ; bioavailability ; degradation ; permeability

Source: Springer Online Journal Archives 1860-2000

Topics: Chemistry and Pharmacology

Notes: Abstract In this study the intestinal degradation and absorption of a synthetic pentapeptide, metkephamid, were investigated in the rat by determination of its wall permeabilities in the small and large intestine and the extent and mechanism of its intestinal degradation. The peptide was metabolized in the gut wall through contact with membrane-bound enzymes in the brush border membrane. The extent of metabolic inactivation depended on the intestinal segment investigated and decreased in the axial direction. No metabolism was found in the colon. The dimensionless wall permeabilities (P w*), determined by single-pass perfusion, were also site dependent. P w* was highest in the ileum [1.91 ± 0.24, (SE); n = 4], followed by the jejunum (1.64 ± 0.34; n = 4) and the colon (0.67 ± 0.38; n = 4). Based on the permeability data alone and under the assumption of no presystemic metabolism, complete bioavailability would be predicted for metkephamid. However, following oral administration, the mean absolute bioavailability was only 0.22 ± 0.065% (n = 3), indicating the overall dominance of degradation in the absorption process. Thus future strategies in oral peptide delivery should focus on increasing the stability of the peptide in the intestine by modifying the peptide structure and/or delivering the compound to an intestinal segment showing little or no enzymatic degradation.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1023/A:1018962415287

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6

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Improving Stability and Release Kinetics of Microencapsulated Tetanus Toxoid by Co-Encapsulation of Additives (1998)

Johansen, Pål ; Men, Ying ; Audran, Régine ; [et al.]

Springer

Pharmaceutical research 15 (1998), S. 1103-1110

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ISSN: 1573-904X

Keywords: antigen delivery ; PLGA microspheres ; tetanus toxoid ; antigenic stability ; stabilizing additives

Source: Springer Online Journal Archives 1860-2000

Topics: Chemistry and Pharmacology

Notes: Abstract Purpose. Tetanus toxoid (Ttxd) encapsulated in polyester microspheres (MS) for single injection immunization have so far given pulsatile in vitro release and strong immune response in animals, but no boosting effect. This has been ascribed to insufficient toxoid stability within the MS exposed to in vivo conditions over a prolonged time period. This study examined the effect of co-encapsulated putative stabilizing additives. Methods. Two different Ttxd were encapsulated in poly(D,L-lactic-co-glycolic acid) (PLGA 50:50) and poly(D,L-lactic acid) (PLA) MS by spray-drying. The influence of co-encapsulated additives on toxoid stability, loading in and release from the MS, was studied by fluorimetry and ELISA. Results. Co-encapsulated albumin, trehalose and γ-hydroxypropyl cyclodextrin all improved the toxoid encapsulation efficiency in PLGA 50:50 MS. Albumin increased the encapsulation efficiency of antigenic Ttxd by one to two orders of magnitude. Further, with albumin or a mixture of albumin and trehalose ELISA responsive Ttxd was released over 1−2 months following a pulsatile pattern. Conclusions. Optimized Ttxd containing MS may be valuable for a single-dose vaccine delivery system.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1023/A:1011998615267

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7

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Chemical Degradation Kinetics of Recombinant Hirudin (HVI) in Aqueous Solution: Effect of pH (1998)

Gietz, Ursula ; Alder, Ruth ; Langguth, Peter ; [et al.]

Springer

Pharmaceutical research 15 (1998), S. 1456-1462

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ISSN: 1573-904X

Keywords: recombinant hirudin ; chemical stability ; protein stability ; succinimide formation ; isomerization ; cyclic imide formation ; capillary electrophoresis ; circular dichroism

Source: Springer Online Journal Archives 1860-2000

Topics: Chemistry and Pharmacology

Notes: Abstract Purpose. To gain information on the chemical stability pattern and the kinetics of the degradation of recombinant hirudin variant HVI (rHir), a thrombin-specific inhibitor protein of 65 amino acids, in aqueous solution as a function of pH. Methods. Stability of rHir was monitored at 50°C in the framework of a classical pH-stability study in aqueous buffers pH 1−9.5. Two capillary electrophoresis (CE) protocols were used; one for the kinetics of succinimide formation at Asp53-Gly54 (C-terminal tail) and Asp33-Gly34 (loop section), the other for the kinetics of rHir degradation. To check for potential effects of conformational changes by thermal denaturation, circular dichroism (CD) measurements were performed between 25 and 80°C. Results. Throughout the pH range studied no effect of thermal denaturation on rHir confirmation at 50°C was observed. rHir was most stable at a neutral pH whereas, at slightly acidic pH, an intermediate stability plateau was found. Both, strongly acidic and alkaline conditions led to fast rHir degradation. Depending on the pH of degradation, rHir was found to degrade in various combinations of multiple parallel and sequential degradation patterns. Special focus was on succinimide formation at Asp53-Gly54 (C-terminal tail) and Asp33-Gly34 (loop) and on the potential of isoAsp formation in position 53 and 33. Conclusions. Chemical rHir stability in the intermediate pH range depends strongly on succinimide formation. At slightly acidic conditions succinimides represent the major degradation product (up to 40%). Around neutral pH succinimides react further, presumably by isoAsp formation, and concentrations remain low. Relative preference of succinimide formation in the C-terminal tail domain versus the loop domain is explained by higher backbone flexibility in the tail.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1023/A:1011918108849

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8

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The Adjuvant Effect of Bacitracin on Nasal Absorption of Gonadorelin and Buserelin in Rats (1988)

Raehs, Susanne C. ; Sandow, Jürgen ; Wirth, Klaus ; [et al.]

Springer

Pharmaceutical research 5 (1988), S. 689-693

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ISSN: 1573-904X

Keywords: nasal absorption ; absorption adjuvants ; bacitracin ; sodium taurodihydrofusidate ; absorption enhancers ; gonadorelin ; buserelin ; mucosal tolerance

Source: Springer Online Journal Archives 1860-2000

Topics: Chemistry and Pharmacology

Notes: Abstract Nasal absorption of gonadorelin (luteinizing hormone-releasing hormone; LH-RH) and buserelin, an LH-RH agonist, was studied in anesthetized rats. Administration of peptides was by nasal instillation of aqueous peptide/buffer solutions. Peptide absorption was monitored using different techniques:(a) by specific radioimmunoassays for serum levels of lutropin (LH), (b) by the cumulative urinary excretion of buserelin, and (c) by the ovulatory activity after nasal LH-RH and buserelin, respectively. Without adjuvant the nasal absorption of LH-RH and buserelin was relatively poor compared to subcutaneous or intravenous injection. Using absorption adjuvants of different types, e.g., sodium taurodihydrofusidate (STDHF) and bacitracin, marked increases in nasal absorption and, therefore, significant nasal adjuvant activity were found, as demonstrated by an increase in the biological response after nasal administration of the peptides. The mucosal compatibility of bacitracin at the concentrations used for enhancement of absorption was confirmed by an in vitro investigation using isolated gastric mucosa of guinea pigs as a test model.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1023/A:1015947509295

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9

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Nasal Epithelial Permeation of Thymotrinan (TP3) Versus Thymocartin (TP4): Competitive Metabolism and Self-Enhancement (2000)

Schmidt, M. Christiane ; Rubas, Werner ; Merkle, Hans P.

Springer

Pharmaceutical research 17 (2000), S. 222-228

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ISSN: 1573-904X

Keywords: nasal absorption ; peptide absorption ; nasal metabolism ; aminopeptidases

Source: Springer Online Journal Archives 1860-2000

Topics: Chemistry and Pharmacology

Notes: Abstract Purpose. To investigate concentration dependent permeabilities andmetabolism kinetics of thymotrinan (TP3) versus thymocartin (TP4)in nasal epithelium in vitro. Methods. Excised bovine nasal mucosa was used as an in vitro model.Permeabilities were studied in a diffusion chamber, metabolism kineticsin a reflection kinetics set-up. Studies were performed at various TP3and TP4 concentrations. The 3H-mannitol flux was measured to monitorjunctional permeability. Potential Ca2+-complexation was investigatedusing a Ca2+-selective electrode. Results. Permeability of TP3 was negligible at 0.1 and 0.2 mM andincreased drastically above 0.4 mM up to ∼2 × 10−5 cm s−1. In thepresence of 2 mM TP4 the TP3 permeabilites were significantly above(∼4 × 10−5 cm s−1) the level of TP3 without TP4, and TP3 metabolismwas totally inhibited. TP3 and TP4 showed a significant concentrationdependent effect on the permeability of 3H-mannitol. Ahyperosmolarity effect of the peptide solutions was excluded. Transepithelialelectrical resistance (TEER; ∼30 Ω cm2) was unchanged by either TP3 orTP4. At 1 mM TP3 the mucosal-to-serosal permeability was four timeshigher than serosal-to-mucosal, indicating enzyme polarization. Inreflection kinetics studies, TP3 degradation was slightly higher on themucosal than on the serosal side. TP3 and TP4 followed the samenon-linear metabolism kinetics. Conclusions. Increase in permeability at high TP concentrationsinvolves competitive enzyme saturation combined with self-enhancedparacellular permeation.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1023/A:1007529716926

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