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The effect of enzymatic inhibition versus increased paracellular route of transport of vasopressin peptides
313
the monomer to initiation by basic groups, such drugs are loaded into the particles after their production. If such particles are to be utilised in uiuo, an essential requirement is that the particle size distribution (PSD ) is known accurately. Computation of average particle size and polydispersity factors from photon correlation spectroscopy (PCS) data is both simple and fast requiring no a priori knowledge of the distribution. Attempts to extract more information from these two parameters rely upon the assumption that the underlying PSD is already known (e.g. log-normal, Schulz or Pearson) and always obeyed. This approach can result in significant errors if the transformations used are carried out incorrectly or applied inappropriately. Algorithms are available (e.g. routine NNLS) which are capable of extracting the intensity weighted PSD function g(T) from raw PCS data with greater reliability than previously possible, with the user required to supply little or no extra information. Concurrent evaluation of the Mie scattering factors for solid spheres (e.g. routine BHMIE) allow these to be converted to the more familiar particle mass distribution. This requires a knowledge of the refractive index of the particles being measured. The refractive index of poly (ethylcyanoacrylate) (PECA) was determined by measuring the angle at which the reflected p-polarized radiation from a thin film of PECA was zero (incident wavelength 632.8 nm) . The PECA nanoparticles were prepared by stirring 500 ~1 of monomer in 25 ml of filtered dilute hydrochloric acid, pH 1.90 or 2.25, containing 0.3% Tween 20. Eight hours later, the suspensions were transferred to dialysis tubing and dialysed for 7 days against daily changes of 10 litres of dilute HCl. Samples were then taken and diluted with 0.2 pm filtered 0.05 M HCl to a suitable concentration for PCS. Correlation functions at 90” were accumulated on a Malvern K7027 digital correlator. Data analysis by the method of cumulants gave the mean particle size and polydispersity for each sample. Two PECA latices of measured size 88 and 209 nm were selected, mixed in a known mass ratio (5.79 : 1) and correlation functions again recorded at 90”. Data from this mixture were analysed using the above mentioned routines. Resolution of the two peaks of the PSD is achieved and the calculated sizes (86.3,215.6 nm) are in agreement with the individual cumulants estimates. The cumulants estimate of size for the mixture (150.2 nm, with a polydispersity index of 0.172) could be interpreted as a moderately broad unimodal distribution and highlights the uncertainty involved in postulating a PSD from cumulants data alone. Using the measured refractive index of 1.485, the Mie correction factors were applied and the relative areas of the two peaks found to be 5.75: 1. The Mie scattering surface for PECA particles was less undulating than that of particles of a higher refractive index, e.g. polystyrene latex. This facilitates the transformation of intensity to mass distributions, though correlation functions must still be of exceptionally high quality if such a transformation is not to result in artifact peaks, especially at the lower end of the distribution. The methods outlined above can also be used to obtain PSDs of spherical nanocapsules by using the coated sphere form factor correction, if the refractive index of both the core material and the polymer wall are known.
THE EFFECT OF ENZYMATIC INHIBITION VERSUS INCREASED ROUTE OF TRANSPORT OF VASOPRESSIN PEPTIDES Anna-Lena
Ungell and Annike
Andreasson
Department of Drug Delivery Research, Pharmaceutical R&D, AB H&s/e,
The Ussing-chamber
technique
PARACELLUIAR
was evaluated
Miilndal (Sweden)
as an oral absorption
model by studies of the rel-
314
ative effect of inhibition of enzymatic degradation and increased paracellular route of transport of the poorly absorbed vasopressin analogs lysine vasopressin (LVP) and l-desaminocysteine-8-D, arginine vasopressin (DDAVP ) . Stripped ileum and colon epithelia from the rat were prepared for a transport-chamber (modified Ussing-chamber) consisting of two lo-ml chambers with rotors for effective stirring of the test solution. Simultaneous measurement of the electrical parameters (PD and SCC ) and observation by light microscopy revealed intact epithelium with no signs of oedema or cell damage during the experimental time of 180 min. Either LVP or DDAVP (250 @f) was added to the mucosal side of the preparations and samples were withdrawn at different time intervals from both sides. LVP, DDAVP and their metabolites were monitored using HPLC with UV-detection and gradient separation. LVP was degraded into 3 major metabolites at the mucosal side of the ileum preparation. The metabolites were similar to those found from incubation with homogenates of the different tissues. The de~adation of both LVP and DDAVP was very slow in the colon preparations. At the concentration of 250 @fat the mucosai side no intact LVP or metabolites could be detected on the serosal side up to 180 min. DDAVP at the same initial concentration was, however, detectable at a steadystate rate of 33 and 24 pmol/ ( min*cm2) for ileum and colon preparations, respectively. Coaddition of a mixture of protease inhibitors (aprotinin and bestatin) decreased the degradation of LVP and gave a detectable rise on the serosal side. Coaddition with cytochalasin-B to increase the paracellular transport, indeed decreased transepithelial resistance of both ileum and colon giving rise to a multifold increase in concentration of both LVP and DDAVP on the serosal side of the colon preparations, but somewhat less effect was observed in the ileum preparations. The effect of cytochalasinB was instantly reversible at the concentrations used. We conclude that the transport-chamber technique is a suitable tool for studying the relative impo~ance of enzymatic de~adation and low permeability of peptide drugs during permeation of the gastrointestinal membranes. The results also indicate that, apart from inhibition of degradation, opening of the paracellular route reversibly may be necessary to increase the absorption rate of peptides, such as the vasopressin analogues.
CORRELATION J. Nita Cogburn,
OF CACO-2 Matthew
TRANSPORT G. Donovan
WITH
HUMAN
ORAL BIOAVAIlABILlTY
and Charles S. Schasteen
Monsanto Company, Corporate Research and Development
Staff, St. Louis, MO 63 798 (U.S.A.)
Transport across the intestinal epithelium is a major barrier to oral delivery of drugs. Investigation of intestinal transport in in uiuo animal models have been complicated by such variables as lumen contents, mucous layer, transit time and hepatic clearance once the compound of interest is in the systemic circulation. EX viva studies have been problematic due to rapid tissue degradation following removal from the animal. Cell culture models offer the possibility of growing epithelial cells on permeable filters and studying their barrier function and transport pathways without the problems described above. We have established an in uitro model of the human small intestinal absorptive cell with the Caco2 cell line. Transport of impermeable molecules, e.g. mannitol, dextrans, demonstrated that Caco-
the monomer to initiation by basic groups, such drugs are loaded into the particles after their production. If such particles are to be utilised in uiuo, an essential requirement is that the particle size distribution (PSD ) is known accurately. Computation of average particle size and polydispersity factors from photon correlation spectroscopy (PCS) data is both simple and fast requiring no a priori knowledge of the distribution. Attempts to extract more information from these two parameters rely upon the assumption that the underlying PSD is already known (e.g. log-normal, Schulz or Pearson) and always obeyed. This approach can result in significant errors if the transformations used are carried out incorrectly or applied inappropriately. Algorithms are available (e.g. routine NNLS) which are capable of extracting the intensity weighted PSD function g(T) from raw PCS data with greater reliability than previously possible, with the user required to supply little or no extra information. Concurrent evaluation of the Mie scattering factors for solid spheres (e.g. routine BHMIE) allow these to be converted to the more familiar particle mass distribution. This requires a knowledge of the refractive index of the particles being measured. The refractive index of poly (ethylcyanoacrylate) (PECA) was determined by measuring the angle at which the reflected p-polarized radiation from a thin film of PECA was zero (incident wavelength 632.8 nm) . The PECA nanoparticles were prepared by stirring 500 ~1 of monomer in 25 ml of filtered dilute hydrochloric acid, pH 1.90 or 2.25, containing 0.3% Tween 20. Eight hours later, the suspensions were transferred to dialysis tubing and dialysed for 7 days against daily changes of 10 litres of dilute HCl. Samples were then taken and diluted with 0.2 pm filtered 0.05 M HCl to a suitable concentration for PCS. Correlation functions at 90” were accumulated on a Malvern K7027 digital correlator. Data analysis by the method of cumulants gave the mean particle size and polydispersity for each sample. Two PECA latices of measured size 88 and 209 nm were selected, mixed in a known mass ratio (5.79 : 1) and correlation functions again recorded at 90”. Data from this mixture were analysed using the above mentioned routines. Resolution of the two peaks of the PSD is achieved and the calculated sizes (86.3,215.6 nm) are in agreement with the individual cumulants estimates. The cumulants estimate of size for the mixture (150.2 nm, with a polydispersity index of 0.172) could be interpreted as a moderately broad unimodal distribution and highlights the uncertainty involved in postulating a PSD from cumulants data alone. Using the measured refractive index of 1.485, the Mie correction factors were applied and the relative areas of the two peaks found to be 5.75: 1. The Mie scattering surface for PECA particles was less undulating than that of particles of a higher refractive index, e.g. polystyrene latex. This facilitates the transformation of intensity to mass distributions, though correlation functions must still be of exceptionally high quality if such a transformation is not to result in artifact peaks, especially at the lower end of the distribution. The methods outlined above can also be used to obtain PSDs of spherical nanocapsules by using the coated sphere form factor correction, if the refractive index of both the core material and the polymer wall are known.
THE EFFECT OF ENZYMATIC INHIBITION VERSUS INCREASED ROUTE OF TRANSPORT OF VASOPRESSIN PEPTIDES Anna-Lena
Ungell and Annike
Andreasson
Department of Drug Delivery Research, Pharmaceutical R&D, AB H&s/e,
The Ussing-chamber
technique
PARACELLUIAR
was evaluated
Miilndal (Sweden)
as an oral absorption
model by studies of the rel-
314
ative effect of inhibition of enzymatic degradation and increased paracellular route of transport of the poorly absorbed vasopressin analogs lysine vasopressin (LVP) and l-desaminocysteine-8-D, arginine vasopressin (DDAVP ) . Stripped ileum and colon epithelia from the rat were prepared for a transport-chamber (modified Ussing-chamber) consisting of two lo-ml chambers with rotors for effective stirring of the test solution. Simultaneous measurement of the electrical parameters (PD and SCC ) and observation by light microscopy revealed intact epithelium with no signs of oedema or cell damage during the experimental time of 180 min. Either LVP or DDAVP (250 @f) was added to the mucosal side of the preparations and samples were withdrawn at different time intervals from both sides. LVP, DDAVP and their metabolites were monitored using HPLC with UV-detection and gradient separation. LVP was degraded into 3 major metabolites at the mucosal side of the ileum preparation. The metabolites were similar to those found from incubation with homogenates of the different tissues. The de~adation of both LVP and DDAVP was very slow in the colon preparations. At the concentration of 250 @fat the mucosai side no intact LVP or metabolites could be detected on the serosal side up to 180 min. DDAVP at the same initial concentration was, however, detectable at a steadystate rate of 33 and 24 pmol/ ( min*cm2) for ileum and colon preparations, respectively. Coaddition of a mixture of protease inhibitors (aprotinin and bestatin) decreased the degradation of LVP and gave a detectable rise on the serosal side. Coaddition with cytochalasin-B to increase the paracellular transport, indeed decreased transepithelial resistance of both ileum and colon giving rise to a multifold increase in concentration of both LVP and DDAVP on the serosal side of the colon preparations, but somewhat less effect was observed in the ileum preparations. The effect of cytochalasinB was instantly reversible at the concentrations used. We conclude that the transport-chamber technique is a suitable tool for studying the relative impo~ance of enzymatic de~adation and low permeability of peptide drugs during permeation of the gastrointestinal membranes. The results also indicate that, apart from inhibition of degradation, opening of the paracellular route reversibly may be necessary to increase the absorption rate of peptides, such as the vasopressin analogues.
CORRELATION J. Nita Cogburn,
OF CACO-2 Matthew
TRANSPORT G. Donovan
WITH
HUMAN
ORAL BIOAVAIlABILlTY
and Charles S. Schasteen
Monsanto Company, Corporate Research and Development
Staff, St. Louis, MO 63 798 (U.S.A.)
Transport across the intestinal epithelium is a major barrier to oral delivery of drugs. Investigation of intestinal transport in in uiuo animal models have been complicated by such variables as lumen contents, mucous layer, transit time and hepatic clearance once the compound of interest is in the systemic circulation. EX viva studies have been problematic due to rapid tissue degradation following removal from the animal. Cell culture models offer the possibility of growing epithelial cells on permeable filters and studying their barrier function and transport pathways without the problems described above. We have established an in uitro model of the human small intestinal absorptive cell with the Caco2 cell line. Transport of impermeable molecules, e.g. mannitol, dextrans, demonstrated that Caco-