Viscoelastic Properties of Bioadhesive,Chlorhexidine-Containing Semi-Solids for Topical Application to the Oropharynx

Purpose. This study examined the viscoelastic properties of bioadhesive, chlorhexidine-containing semi-solid formulations, designed for topical application to the oropharynx. Methods. Oscillatory rheometry was performed using a Carri-Med CSL²-100 rheometer at 20.0 ± 0.1° C in conjunction with parallel plate geometry (2 cm diameter, 0.5 mm sample thickness). Samples were subjected to a constant strain (6.5 × 10^–3 rad) and defined viscoelastic parameters, namely storage modulus (G), loss modulus (G), loss tangent (tan ) and dynamic viscosity (), measured over a defined frequency range (0.01-1.0 Hz). Results. As the oscillatory frequency was increased, G G of all formulations increased, whereas both and tan significantly decreased. The magnitude of increase of G and G as a function of frequency was relatively small, indicating that, in general, the formulations were non-cross-linked elastic systems. Increasing concentrations of HEC, PVP and PC significantly increased G, G, yet decreased tan observations that may be attributed to the physical state of each polymer in the formulations. Formulation elasticity increased (i.e. tan decreased) as a result of increased entanglement of polymeric chains of dissolved components (i.e. HEC and PVP) and the restrained extension of swollen, cross-linked chains of PC. Additionally, in formulations where the saturation solubility of PVP was exceeded and/or insufficient 'free-water' was available for maximal swelling of PC, formulation elasticity increased as a result of the increasing mass of dispersed solid particles of PVP and/or PC. Formulation increased due to the attendent effects of polymer chain entanglement and polymer state on overall formulation viscosity. Conclusions. Following application to the oropharynx, the formulations will behave as elastic systems. Thus, these formulations would be expected to offer advantageous clinical properties, e.g., prolonged drug release, increased bioadhesion. However, it is noteworthy that the final choice of formulation for clinical evaluation will involve a compromise between viscoelastic characteristics and acceptable textural properties, e.g. ease of product application. This study has shown the applicability of oscillatory rheometry for both the characterisation and selection of candidate, topical bioadhesive formulations for clinical evaluation.     

Evaluation of Cytotoxicity of Various Ophthalmic Drugs, Eye Drop Excipients and Cyclodextrins in an Immortalized Human Corneal Epithelial Cell Line

Purpose. An immortalized human corneal epithelial cell line (HCE) was tested as a screening tool for prediction of topical ocular irritation/ toxicity by pharmaceuticals. Methods. Effects of various drugs, excipients and cyclodextrins (CDs) on viability of HCE cells were evaluated using two in vitrocytotoxicity tests, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye reduction assay and propidium iodide assay. Results. Mitochondrion-based MTT test was a more sensitive indicator of cytotoxicity than the plasma membrane-based propidium iodide test. The tests revealed following cytotoxic rankings for ophthalmic drugs: dipivefrin > timolol > pilocarpine dexamethasone; for excipients: benzalkonium chloride (BAC) > sodium edetate (NA₂EDTA) > poly-vinyl alcohol (PVA) > methylparaben; and for CDs: -CD > dimethyl--cyclodextrin (DM--CD) > sulfobutyl ether (-cyclodextrin ((SBE)_7m--CD) hydroxypropyl--cyclodextrin (HP--CD) > -CD. In consideration of the in vivoclinical situation, the short exposure time (5 min) is more relevant even though toxic effects of some test substances were seen only after longer exposure times (30 and 60 min). Conclusions. Immortalized HCE cells are a promising tool for rapid cytotoxicity assays of ocular medications. The cell line is potentially useful in predicting the in vivocorneal toxicity of ocularly applied compounds.     

β2-Adrenergic Receptor Genotype-Related Changes in cAMP Levels in Peripheral Blood Mononuclear Cells After Multiple-Dose Oral Procaterol

Purpose. To evaluate the ₂-adrenergic receptor (₂AR) genotype frequency in the Japanese population and the relationship between ₂AR genotype at amino acid position 16 (₂AR-16) and desensitization to ₂-agonist ex vivo. Methods. The ₂AR genotypes at amino acid positions 16, 27, and 164 of 92 healthy Japanese subjects were determined by polymerase chain reaction-restriction fragment-length polymorphism. The relationship between the ₂AR-16 genotype and the desensitization to ₂-agonist was examined in 10 male subjects ex vivo. Procaterol tablet (HCl salt, 50g, Meptin®) was given orally for 5 days, and peripheral blood was obtained before and after 5 days of consecutive medications followed by the assessment of the intracellular cAMP levels in peripheral blood mononuclear cells after incubation with or without procaterol hydrochloride (0-1000 ng/mL). Results. Allele frequency was Arg16:Gly16 = 46%:54%, Gln27:Glu27 = 92%:8%, and Thr164:Ile164 = 100%:0%, respectively. The cAMP levels were increased by incubation with procaterol hydrochloride, and the increase was suppressed after 5 days of consecutive medications. The suppression was more significant in the homozygote for Gly16 than the homozygote for Arg16. Conclusions. The desensitization to ₂-agonist was associated more frequently with the mutation at ₂AR-16 (Gly16).     

Transport Characteristics of Peptidomimetics. Effect of the Pyrrolinone Bioisostere on Transport Across Caco-2 Cell Monolayers

Purpose. To compare the permeation characteristics of amide bond-containing HIV-1 protease inhibitors and their pyrrolinone-containing counterparts across Caco-2 cell monolayers, a model of the intestinal mucosa. Methods. Transepithelial transport and cellular uptake of three pairs of amide bond-containing and pyrrolinone-based peptidomimetics were assessed in the presence and absence of cyclosporin A using the Caco-2 cell culture model. The potential of the peptidomimetics to interact with biological membranes was estimated by IAM chromatography. Results. In the absence of cyclosporin A, apical (AP) to basolateral (BL) flux of all compounds studied was less than the flux determined in the opposite direction (i.e., BL-to-AP). The ratio of the apparent permeability coefficients (P_app) calculated for the BL-to-AP and AP-to-BL transport (P_BLAP/P_APBL) varied between 1.7 and 36.2. When individual pairs were compared, P_BLAP/P_APBL ratios of the pyrrolinone-containing compounds were 1.5 to 11.5 times greater than those determined for the amide bond-containing analogs. Addition of 25 M cyclosporin A to the transport buffer reduced the P_BLAP /P_APBL ratios for all protease inhibitors to a value close to unity. Under these conditions, the amide bond-containing peptidomimetics were at least 1.6 to 2.8 times more able to permeate Caco-2 cell monolayers than were the pyrrolinone-containing compounds. The intrinsic uptake characteristics into Caco-2 cells determined in the presence of 25 M cyclosporin A were slightly greater for the amide bond-containing protease inhibitors than for the pyrrolinone-containing analogs. These uptake results are consistent with the transepithelial transport results determined across this in vitro model of the intestinal mucosa. Conclusions. The amide bond-containing and pyrrolinone-based peptidomimetics are substrates for apically polarized efflux systems present in Caco-2 cell monolayers. The intrinsic permeabilities of the amide bond-containing protease inhibitors are slightly greater than the intrinsic permeabilities of the pyrrolinone-based analogs through Caco-2 cell monolayers.     

NMR Studies of Retinoid-Protein Interactions: The Conformation of [13C]-β-Ionones Bound to β-Lactoglobulin B

Purpose. Vitamin A (retinol) and its metabolites comprise the natural retinoids. While the biological action of these molecules are thought to be primarily mediated by ca. 55 kDa nuclear retinoic acid receptors, a number of structurally similar 15-20 kDa proteins are involved in the transport, and possibly metabolism, of these compounds. The milk protein -lactoglobulin B (-LG) is an 18 kDa protein which binds retinol and may be involved in oral delivery of retinol to neonates. -LG also binds drugs and other natural products and is of potential interest as a protective delivery vehicle. Methods. To examine the conformation of the model retinoid -ionone both in solution and when bound to -LG, NMR and computational methods have been employed. Results. Taken together, NMR studies of -ionone in solution measuring scalar and dipolar coupling, as well as CHARMm calculations, suggest -ionone prefers a slightly twisted 6-s-cis conformation. Isotope-edited NMR studies of ^l3C-labeled -ionones bound to -LG, primarily employing the HMQC-NOE experiment, suggest -ionone also binds to -LG in its 6-s-cis conformation. Conclusions. The methods employed here allow estimates of protein-bound ligand conformation. However, additional sites of ligand labeling will be necessary to aid in binding site localization.     

A Simple Rheological Method for the in Vitro Assessment of Mucin-Polymer Bioadhesive Bond Strength

A simple viscometric method was used to quantify mucin-polymer bioadhesive bond strength. Viscosities of 15% (w/v) porcine gastric mucin dispersions in 0.1 N HC1 (pH 1) or 0.1 N acetate buffer (pH 5.5) were measured with a Brookfield viscometer in the absence (_m) or presence (_t) of selected neutral, anionic, and cationic polymers (0.1–2.5%, w/v). Viscosity components of bioadhesion (1%) were calculated from the equation, _t = _m + _p + _b, where _p is the viscosity of corresponding pure polymer solution as measured by an Ostwald viscometer. The forces of bioadhesion (F) were calculated from the equation, F = _b, where is the rate of shear/sec. _b's and F's for polyelectrolytes, e.g., polyacrylic acid, cationic gelatin, and chitosan were always higher in acetate buffer than in HC1. Validity of the technique and the effect of ionic charge, polymer conformation, and rate of shear on _b and F are discussed, as is a comparison of this method to other methods for evaluating bioadhesive materials.     

Effect of Cyclodextrin Charge on Complexation of Neutral and Charged Substrates: Comparison of (SBE)7M-β-CD to HP-β-CD

Purpose. To understand the role of charge in substrate/cyclodextrin complexation by comparing the binding of neutral and charged substrates to a neutral cyclodextrin, such as hydroxypropyl –CD (HP––CD) with 3.5 degrees of substitution, and an anionically charged cyclodextrin, such as sulfobutyl ether –CD ((SBE)_7M––CD) with 6.8 degrees of substitution. Method. HP––CD and (SBE)_7M––CD were evaluated in their ability to form inclusion complexes with neutral compounds, as well as to cationic and anionic substrates in their charged and uncharged forms. The complexation constants (K_c) were determined via a UV spectrophotometric technique, by monitoring the change in substrate absorbance upon incremental addition of a concentrated cyclodextrin solution. The role of electrostatic interaction was probed by observing K_c as a function of solution ionic strength. Results. Neutral molecules displayed a stronger interaction with (SBE)_7M––CD compared to HP––CD. In those cases where the guest possessed a charge (positive or negative), HP––CD/substrate complexes exhibited a decrease in complexation strength (2 to 31 times lower) compared to the neutral forms of the same substrate. The same was true (but to a larger extent, 41 times lower) for negatively charged molecules binding to (SBE)_7M––CD due to charge–charge repulsion. However, positively charged molecules interacting with the negatively charged (SBE)_7M––CD displayed a similar binding capability as their neutral counterpart, due to charge–charge attraction. Further evaluation through manipulation of solution ionic strength revealed strong electrostatic interactions between substrate and cyclodextrin charges. In addition, the studies suggested that on average two sulfonates out of seven may be involved in forming ionic attraction or repulsion effects with the positive charges on prazosin and papaverine, or negative charges of ionized naproxen and warfarin. Conclusions. Presence of charge on the cyclodextrin structure provides an additional site of interaction compared to neutral cyclodextrins, which may be modified using solution ionic strength.     

Development and Validation of Two Solid-Phase Enzyme Immunoassays (ELISA) for Quantitation of Human Epidermal Growth Factors (hEGFs)

Purpose. The purpose of the present investigation was to develop and validate two separate enzyme-linked immunosorbent assays (ELISA) for quantitation of exogenous human epidermal growth factor (hEGFl -53) and its truncated fragment (hEGFl-48) in rat plasma. Methods. The present assay systems were based on the sandwiching of the antigen between a monoclonal mouse anti-hEGFl-53 antibody, pre-coated on a 96-well polystyrene plate, and a polyclonal rabbit anti-hEGFl-48 antibody, which is then detected with a peroxidase-labeled goat anti-rabbit antibody. Results. The calibration curves for hEGFl-48 and hEGFl -53 in plasma were validated over a concentration range of 7.8–250 and 62.5–1000 pg/ml, respectively. Determined from replicate assays of hEGFl-48 quality control samples, the intra-assay precision and accuracy were 8.8% RSD and within ± 9.8%; and the inter-assay precision and accuracy were 14.8% RSD and within ± 9.7% RE, respectively. Determined from replicate assays of hEGFl-53 quality control samples, the intra-assay precision and accuracy were 10.0% RSD and within ± 8.5%; and the inter-assay precision and accuracy were 10.0% RSD and within ± 5.7% RE, respectively. The limit of quantitation of the hEGFl-48 and hEGFl-53 assay using 200 µL plasma per well is 7.8 and 62.5 pg/ml, respectively. These two ELISA methods are specific to hEGFs and do not cross-react with mouse EGF or other growth factors (TGF, TGF, PDGF, and FGF) or lymphokines (IL₁ and TNF). These validated methods have been routinely applied to assay of plasma samples from various pharmacokinetic studies in rats receiving intravenous hEGFs. Both assay methods were also adapted to assay endogenous hEGFs in biological fluids of different animal species. Conclusions. Two sensitive ELISA methods have been validated for quantitation of hEGFl–53 and hEGFl–48 in rat plasma. Their utility has been demonstrated in the application of assaying immunoreactive concentrations of exogenous and endogenous epidermal growth factors.     

Dose-response relationship of the β-adrenoceptor antagonist bisoprolol in patients with coronary heart disease and chronic obstructive bronchitis

Summary The effects of single, consecutively increased, oral doses of the -adrenoceptor antagonist bisoprolol (5, 10, 15, 20, 30 and 40 mg) on blood pressure, heart rate and bronchomotor tone were investigated in an open acute trial with 16 patients suffering from angina pectoris due to coronary heart disease and reversible chronic obstructive bronchitis. Even the lowest dose of bisoprolol (5 mg) caused a marked, long-lasting reduction in blood pressure and heart rate. After doses exceeding 20 mg, the incidence of an exaggerated pharmacodynamic effect on heart rate ( ₁-blockade) increased with dose. At doses above 30 mg, bisoprolol showed incipient impairment of bronchomotor function ( ₂-blockade) in individual patients. It is concluded that bisoprolol exhibits high ₁-selectivity, i.e. a wide ₁/ ₂ split, since blockade of bronchial ₂-receptors only occurred at doses well above the therapeutically relevant dose range. The results may not be applicable to chronic treatment.     

Analysis of the Initial Burst of Drug Release Coupled with Polymer Surface Degradation

Purpose. Local pH effect on the release of a model pH-inert hydrophobic drug coupled with polymer degradation is described at the induction phase of biodegradable polymer erosion for better understanding the nature of initial burst of a drug. Methods. Using a novel approach with time-of-flight secondary ion mass spectrometry, both surface concentration of Ph₃N and degradation kinetics of PLLA are simultaneously and independently determined from a model Ph₃N/PLLA (20:80 wt%) blend matrix (t 0.4 m on 1.0 cm²). In vitro hydrolysis of the model blend matrix is investigated for short-term periods (<24 h) at physiologic pH and temperature and compared to basic pH. Results. The rate of PLLA degradation is accelerated by a factor of 3 when using basic pH in vitro, but the rate of Ph₃N accumulation at the surface is accelerated by a factor of 6. Conclusions. A new quantitative method has been developed to examine the earliest stages of polymer degradation and drug release. It was applied to a model system that could not be examined by traditional in vitro methods. For the model system studied the release of a low molecular weight hydrophobic drug at the induction phase of polymer erosion is related to but not singularly dependent on degradation kinetics.     

Inactivation and Aggregation of β-Galactosidase in Lyophilized Formulation Described by Kohlrausch-Williams-Watts Stretched Exponential Function

Purpose. To examine whether the empirical Kohlrausch-Williams-Watts (KWW) equation is applicable not only to protein aggregation but also to protein denaturation in lyophilized formulations. Lyophilized -galactosidase (-GA) formulations containing polyvinylalcohol and methylcellulose were used as model formulations. The possibility of predicting storage stability based on the temperature dependence of the estimated parameters of inactivation/aggregation—time constant () and its distribution () is discussed. Methods. Protein aggregation in lyophilized -GA formulations at 10-70°C and 6-43% relative humidity was determined as a function of time by size exclusion chromatography. Enzyme activity was also determined using 2-nitrophenyl--D-galactopyranoside as a substrate. Results. Inactivation and aggregation of -GA were describable with the empirical KWW equation, regardless of whether the temperature was above or below the NMR relaxation-based critical mobility temperature (T_mc) or whether protein molecules with different degrees of deformation resulting from stresses during lyophilization exist in the formulation. The estimated parameter for protein aggregation decreased rapidly as temperature increased beyond T_mc because the mobility of polymer molecules increased in the initial stages of glass transition. The time required for 10% enzyme to aggregate (t₉₀) calculated from the and parameters exhibited a change in temperature dependence gradient near T_mc. In contrast, t₉₀ for protein inactivation exhibited temperature dependence patterns varying with the excipients. Conclusions. The t₉₀ calculated from the estimated and parameters was found to be a useful parameter for evaluating the stability of lyophilized -GA formulations. The prediction of t₉₀ by extrapolation was possible in the temperature range in which did not rapidly vary with temperature.     

Biliary Excretion of 17β-Estradiol 17β-d-Glucuronide Is Predominantly Mediated by cMOAT/MRP2

Purpose. The mechanism for the biliary excretion of 17-estradiol170-d-glucuronide (E₂17G), a cholestatic metabolite of estradiol, isstill controversial. The purpose of the present study is to examine thetransport of E₂17G across the bile canalicular membrane. Methods. We examined the uptake of [³H]E₂17G by isolatedcanalicular membrane vesicles (CMVs) prepared from Sprague-Dawley (SD)rats and Eisai Hyperbilirubinemic rats (EHBR) whose canalicularmultispecific organic anion transporter/multidrug resistance associatedprotein 2 (cMOAT/MRP2) function is hereditarily defective. Also,in vivo biliary excretion of intravenously administered [³H]E₂17Gwas examined. Results. In CMVs prepared from SD rats, but not from EHBR, amarked ATP-dependent uptake of [³H]E₂17G was observed.Moreover, E₂17G competitively inhibited the ATP-dependent uptake of[³H]2,4-dinitrophenyl-S-glutathione (DNP-SG). In addition, nosignificant inhibitory effect of verapamil (100 M) and PSC-833 (5 M) onthe uptake of [³H]E₂17G was observed. In vivo, the biliary excretionof intravenously administered [³H]E₂17G was severely impaired inEHBR while the biliary excretion of [³H]E₂17G in SD rats wasreduced by administering a cholestatic dose (10 mol/kg) unlabeledE₂17G, but not by PSC-833 (3 mg/kg). Conclusions. The transport of E₂17G across the bile canalicularmembrane is predominantly mediated by cMOAT/MRP2.     

Intradermal Injection of Transforming Growth Factor-β1 Gene Enhances Wound Healing in Genetically Diabetic Mice

Purpose. To evaluate the biologic effect of direct cutaneous TGF-1 gene delivery on impaired wound healing models using genetically diabetic mice. Methods. Diabetic mice (C57BKS.Cg-m +/+ Leprdb female mice) with 1 cm × 1 cm excisional wounds were intradermally injected with 60 g of plasmid DNA encoding TGF-1 gene. The wound closure was measured up to 14 days postwounding. At days 7 and 14 postwounding, sections of skin were taken for hematoxylin and eosin and Masson's trichome staining to examine the morphology and collagen deposition. The cell proliferation and TGF-1 gene expression were studied using immunohistochemical stainings for 5-bromo-2-deoxy-uridine and for TGF-1. Results. A higher cell proliferation rate and a denser and more organized new extracellular matrix were observed in the treated wound site. Complete wound closure was detected as early as 7 days for TGF-1-treated group in comparison with 11-14 days for the untreated, control plasmid DNA- and PBS-treated groups. Conclusion. A single intradermal injection of TGF-1 plasmid DNA was sufficient to enhance wound healing. This approach represents a new strategy that may be applied to the treatment of excisional wounds in human diabetic patients.     

Targeting the sodium-dependent multivitamin transporter (SMVT) for improving the oral absorption properties of a retro-inverso Tat nonapeptide

Purpose. To investigate the potential for delivering large peptides orally by altering their absorptive transport pathways and improving intestinal permeability. The absorptive transport of retro-inverso (R.I.-) K-Tat9 and R.I.-K(biotin)-Tat9, novel peptidic inhibitors of the Tat protein of HIV-1, and their interactions with human SMVT (hSMVT), a high affinity, low capacity transporter, were investigated using Caco-2 and transfected CHO cells. Methods. Following synthesis on a PAL resin using Fmoc chemistry, the transport of R.I.-K-Tat9 (0.01-25 M) and R.I.-K(biotin)-Tat9 (0.1-25 M) was evaluated across Caco-2 cells. The transport and kinetics of biotin, biocytin and desthiobiotin (positive controls for SMVT) were also determined. Uptake of R.I.-K-Tat9 and R.I.-K(biotin)-Tat9 (both 0.1-10 M) was determined in CHO/hSMVT and CHO/pSPORT (control) cells. Results. The absorptive transport of R.I.-K-Tat9 was passive, low (P_m1 × 10^–6 cm/sec) and not concentration dependent. R.I.-K(biotin)-Tat9 permeability was 3.2-fold higher than R.I.-K-Tat9 demonstrating active (E_a = 9.1 kcal/mole), concentration dependent and saturable transport (K_m = 3.3 M). R.I.-K(biotin)-Tat9 uptake in CHO/hSMVT cells (K_m = 1.0 M) was 500-fold greater than R.I.-K-Tat9 (at 10 M). R.I.-K(biotin)-Tat9 transport in Caco-2 and CHO/hSMVT cells was significantly inhibited by known substrates of SMVT including biotin, biocytin, and desthiobiotin. Passive uptake of R.I.-K(biotin)-Tat9 was significantly greater than R.I.-K-Tat9 uptake in CHO/pSPORT cells. Conclusions. These results demonstrate that the structural modification of R.I.-K-Tat9 to R.I.-K(biotin)-Tat9 altered its intestinal transport pathway resulting in a significant improvement in its absorptive permeability by enhancing nonspecific passive and carrier-mediated uptake by means of SMVT. The specific interactions between R.I.-K(biotin)-Tat9 and SMVT suggest that targeting approaches utilizing transporters such as SMVT may substantially improve the oral delivery of large peptides.     

Restricted Intestinal Absorption of Some β-Lactam Antibiotics by an Energy-Dependent Efflux System in Rat Intestine

Purpose. The purpose of this study was to examine factors limiting the intestinal absorption of orally inactive -lactam antibiotics. Methods. Permeation behaviors of various -lactam antibiotics across rat intestinal segments were evaluated in vitro using diffusion cells. Results. Poorly absorbed -lactam antibiotics, like cephaloridine and cefoperazone, commonly exhibit greater serosal-to-mucosal permeation than mucosal-to-serosal permeation, while cephalexin permeation was greater in the mucosal-to-serosal direction. In the absence of D-glucose, secretory-oriented permeation of cephaloridine and cefoperazone disappeared. Addition of sodium azide into an experimental buffer including D-glucose significantly and selectively enhanced mucosal-to-serosal permeation of cephaloridine and cefoperazone. Although benzylpenicillin, ampicillin, and amoxicillin all showed secretory-oriented permeation, the tendency to permeation was greatest with benzylpenicillin and least with amoxicillin. Probenecid stimulated mucosal-to-serosal permeation of cephaloridine, but verapamil and p-aminohippuric acid had no significant effect on it. Conclusions. It has been suggested that mechanisms which induce secretory-oriented permeation of orally inactive -lactam antibiotics are factors limiting intestinal absorption of such antibiotics. This energy-demanding efflux system was distinct from P-glycoprotein-mediated transport. A free -amino group in the molecule is an important factor for reducing an affinity with the efflux system.     

The Influence of Sodium Glycocholate and Other Additives on the in vivo Transfection of Plasmid DNA in the Lungs

Purpose. A plasmid containing the luciferase marker cDNA was constructed to test non viral gene delivery formulations in vivo. Methods. A scale up procedure was devised to produce up to gram quantities of plasmid. Sufficient quantities were generated to process and test the DNA with various additives and to generate a spray-dried powder formulation of the plasmid. Male Sprague-Dawley rats (250 g) were intratracheally instilled with 200–250 µl of solution containing 200 µg plasmid ± lipid [DC Chol:DOPE 1:1 molar (2mg/kg)] growth factors [KGF (10 mg/kg), EGF (5 mg/kg)], permeation enhancers [sodium glycocholate (0.01 to 10% w/v)), sodium deoxycholate (1% w/v), beta-cyclodextrin (1% w/v)], surfactant [Tween 80 (1% w/v)], a mucolytic [N-acetylcysteine (10% w/v)] and positively charged synthetic polymers [PVAVAM 6 and 14%]. Animals were sacrificed 24 hr post-dose and the lungs were assayed for luciferase using a chemiluminescent assay. Results. The relative ability of the materials to promote luciferase production in the lungs was permeation enhancer >> DNA alone lipid, mucolytic, surfactant, growth factor > polymer. Protein production in the lungs ranged from 10 times below the DNA control (16 pg) using the polymers (1.5 pg) to 125 times greater than the control using the permeation enhancer (2050 pg). The transfection capabilities of the majority of additives was low. The enhancing effects of sodium glycocholate were dose-dependent and perhaps associated with the critical micelle concentration. Although the bile salt was the most successful of the tested compounds, it resulted in significant mortality when used at concentrations greater than 1 % w/v. Conclusions. The results suggest that transfection can be significantly enhanced by additives such as NaGC but some toxicity may be unavoidable.     

Usefulness of the Kohlrausch-Williams-Watts Stretched Exponential Function to Describe Protein Aggregation in Lyophilized Formulations and the Temperature Dependence Near the Glass Transition Temperature

Purpose. We studied the feasibility of using the Kohlrausch-Williams-Watts stretched exponential function (KWW equation) to describe protein aggregation in lyophilized formulations during storage. Parameters representing mean aggregation time (_a) and stretched exponential constant (_a) were calculated according to the KWW equation by assuming that the time required for protein molecules to aggregate () varies because of the fact that protein aggregation occurs at a rate that depends on the degree of protein deformation resulting from stresses created during freeze-drying. The temperature dependence of the parameters near the glass transition temperature was examined to discuss the possibility of predicting protein aggregation by accelerated testing. Methods. Protein aggregation in lyophilized bovine serum -globulin (BGG) formulations containing dextran or methylcellulose, at temperatures ranging from 10 to 80°C, was followed by size-exclusion chromatography. Results. Non-exponential BGG aggregation in lyophilized formulations could be described by the KWW equation. The _a and _a parameters changed abruptly around the NMR relaxation-based critical mobility temperature for formulations containing dextran and methylcellulose. In the glassy state, in contrast, the _a parameter of these formulations exhibited continuous temperature dependence. The parameter , as calculated from _a and _a, reflected differences in values between the two excipients. Conclusions. The results indicate that the parameter _a is reflective of physical changes wihtin lyophilized formulations. Within the temperature range, during which no abrupt changes in _a were observed, knowledge regarding the _aand _a parameters allows the rate of protein aggregation to be predicted. The parameter was found to be useful in comparing the protein aggregation behavior of formulations having different _a and _a values.     

Extensive Biliary Excretion of the Model Opioid Peptide [D-PEN2,5] Enkephalin in Rats

Purpose. This study was designed to test the hypothesis that the enzymatically stable opioid peptide, [D-pen^2,5] enkephalin (DPDPE), is excreted extensively into bile. Methods. Following an i.v. bolus dose of DPDPE (10 mg/kg) to rats, concentrations of DPDPE in serum, bile, liver homogenate and urine were measured by a novel capillary zone electrophoresis method. Data were analyzed to recover the fundamental pharmacokinetic parameters (volumes of distribution; distribution and elimination rate constants governing DPDPE systemic and biliary disposition). Parallel in vitro experiments were performed to evaluate the partitioning of DPDPE between erythrocytes and plasma, as well as to assess the degree of binding of DPDPE to serum proteins. Results. The majority of the administered dose (~80%) was recovered from bile as intact peptide. DPDPE disposition was best described by a two-compartment model with Michaelis-Menten elimination (K_m: 37.5 ± 11 g/ml; V_max: 1143 ± 368 g/min/kg) from the central compartment into bile, suggestive of an active hepatic transport system. DPDPE was associated with a distributional space of 486 ± 62 ml/kg. In vitro incubation of DPDPE with whole blood showed that ~65% of the peptide was associated with erythrocytes. The difference between concentrations of DPDPE in erythrocytes and plasma was statistically significant (29.2 ± 4.9 vs. 18.1 ± 3.1 g/ml, p < 0.05), but not between whole blood and plasma (21.3 ± 2.8 vs. 18.1 ± 3.1 g/ml, p > 0.05). Concentration-independent binding of DPDPE to serum proteins was evidenced between 10 and 100 (g/ml, with an unbound fraction of 0.517 ± 0.182. Conclusions. DPDPE undergoes extensive biliary excretion after i.v administration in rats. The apparent nonlinearity in the biliary excretion of DPDPE revealed by the pharmacokinetic modeling strongly suggests the existence of an active transport system(s) in hepatocytes which may mediate the rapid disappearance of DPDPE from the systemic circulation.     

Comparison of the densities of 5-HT4 receptors, β1- and β2-adrenoceptors in human atrium: functional implications

We measured in human atrium the density of 5-HT₄ receptors, labelled with [¹²⁵I]-SB 207710 (1-butyl-4-piperidinyl) methyl 8-amino-7-iodo-1, (4-benzodioxan-5-carboxylate), and compared it with the density of ₁- and ₂-adrenoceptors, labelled with (–)-[¹²⁵I]-cyanopindolol. [¹²⁵I]-SB 207710 (5–1200 pmol/l) labelled a small population of saturable binding sites (B _max 4 fmol/mg protein) with a pK_D of 9.7 and with 5-HT₄ receptor characteristics, as assessed with competing ligands. The density of atrial binding sites with 5-HT₄ receptor characteristics was 10 and 5 times lower, respectively, than the density of ₁- and ₂-adrenoceptors. We suggest that the small 5-HT₄ receptor population may in part explain why the positive inotropic effects of 5-HT are smaller than those of catecholamines mediated through ₁- and ₂-adrenoceptors.     

Agonist-antagonist properties of fluorescent opioid probes in the guinea-pig myenteric plexus-longitudinal muscle preparation

Summary The behavioral consequences of -adrenoceptor subsensitivity were investigated by determining whether a physiological response that is mediated by -receptors, isoproterenol-induced drinking (IID), would be reduced by subacute antidepressant/ ₂-antagonist treatmentThe coadministration of typical (e.g., imipramine) or atypical (e.g., mianserin) antidepressants with yohimbine or piperoxan twice daily for four consecutive days reduced IID. Both the time course as well as the magnitude of -adrenoceptor subsensitivity could be behaviorally demonstrated. In addition, the reduction in IID observed after coadministration of imipramine with yohimbine was a centrally mediated effect since it was observed after systemic (subcutaneous) and central (intraventricular) administration of isoproterenol. These results provide evidence that IID is an appropriate behavioral model to demonstrate -adrenoceptor subsensitivity following subacute antidepressant/ ₂-antagonist treatment.Paper presented in part at FASEB, St. Louis, MO, USA (Abstract: Fedn Proc 43:941, 1984)