Pharmaceutical Research

Theis, Don; Lucisano, Leo; Halstead, Gordon

doi: 10.1023/A:1018912210202pmid: 7971703

Certain delivery systems are intended to release the active ingredient in different phases to obtain the desired therapeutic effect. For these formulations, such as a bilayer tablet, it is desirable to distinguish and measure the release of drug from the different phases simultaneously. Mass spectrometric methods were developed to measure three ibuprofen isotopomers in serum and two in dissolution fluid. The analytical methods were linear (r ≥ 0.992) over the concentration range of interest and recovery was greater than 99.2% for all isotopomers. Coadministration of [2H0]ibuprofen, [2H4]ibuprofen, and [2H7]ibuprofen to male beagles demonstrated that the isotopomers were bioequivalent and verified the absence of any kinetic isotope effect due to deuterium incorporation (p = 0.286). These methods were then used to evaluate a bilayer tablet formulation composed of an immediate release layer of 100 mg [2H4]ibuprofen and a sustained release layer with a drug load of 300 mg [2H0]ibuprofen. Two different rate-controlling polymer matrices that provided similar in vitro dissolution profiles were compared in the sustained release phase, while the immediate release formulation remained the same. In male beagles, the HPMC matrix delivered a significantly greater amount of ibuprofen (p < 0.05). The AUC was threefold greater for HPMC (1067 ± 437 nmole * h/ml) versus EUDRAGIT® (320 ± 51), and Cmax was nearly four times greater (145 ± 62.1 nmole/ml for HPMC versus 37.9 ± 14.4 for EUDRAGIT®). Although Tmax for HPMC (3.4 ± 1.9 h) lagged behind EUDRAGIT® (2.0 ± 0.82 h), the difference was not significant (p > 0.05). The immediate release layer was absorbed to the same extent as an oral solution (containing [2H7]ibuprofen) that was administered concomitantly with the bilayer tablet. Using the stable isotope markers also demonstrated that the release rates of the two layers were independent of each other, both in vivo and in vitro. Stable isotope techniques are a useful tool in the development of biphasic release formulations since they can be used to determine proper drug load of each phase as well as the appropriate rate of release.

Teraoka, Reiko; Otsuka, Makoto; Matsuda, Yoshihisa

doi: 10.1023/A:1018964227041pmid: 7971704

The effect of ultraviolet (UV) or visible light (VIS) irradiation on the chemical stability of ethyl icosapentate [ethyl-(all-cis)-5,8,l l,14,17-icosapentaenoate] (EPA) was investigated at 45°C by means of HPLC and by measuring the peroxide value (POV). EPA was oxidized to peroxides after an induction period by photoirradiation, and the peroxide subsequently degraded to secondary products. The autoxidation of EPA followed consecutive reaction kinetics including an induction period, and the kinetic parameters of the oxidation were calculated based upon the consecutive reaction model by computer curve fitting. The results of the degradation rate constant, k, and the induction period obtained by HPLC showed that the radical and the peroxide formation rates are affected by UV, but not by VIS light irradiation. The formation rate constant of peroxide, kl, and its degradation rate constant to secondary products, k2, obtained from the POV under UV light irradiation, increased with irradiation intensity, during which the induction period decreased. On the other hand, kl, k2 and the induction period by VIS light irradiation did not change significantly. The relationship between the induction periods obtained by HPLC and POV and the UV light irradiation energy were superimposed in the plots, indicating that these parameters depended on the UV irradiation energy. The relationship between kl/k2 ratio and the UV irradiation energy suggested that the formation of secondary products was more remarkably accelerated by UV energy than that of peroxide.

Delie, Florence; Couvreur, Patrick; Nisato, Dino; Michel, Jean-Baptiste; Puisieux, Francis; Letourneux, Yves

doi: 10.1023/A:1018916311111pmid: 7971705

A diglyceride derivative of a pentapeptide renin inhibitor, the 1,3-dipalmitoyl-[Iva-Phe-Nle-Sta-Ala-Sta-acetyl]-glycerol was synthesized and tested in vitro as a potential prodrug for oral administration. The ability of the diglyceride analog to inhibit the renin activity was equivalent to that of the parent peptide after predigestion with pancreatic lipase. Furthermore, the presence of the palmitoyl groups was found to induce, in vitro, an efficient protection of the peptide from gastric and intestinal hydrolysis. During incubation with intestinal and gastric fluids, and with α-chymotrypsin and pancreatic lipase, the glycerolipidic derivative was more stable than the peptide alone. These results support the use of glycerolipidic prodrug for oral administration of peptides.

Rao, Renée; Lefebvre, Frédéric; Commenges, Gérard; Castet, Catherine; Miguel, Lygie; Maire, Gérard; Gachon, Michel

doi: 10.1023/A:1018968327949pmid: 7971706

The comparison of 13C-NMR spectra of Amiodarone · HC1 in different solvents with the CPMAS spectrum and the crystal structure shows that the molecule adopts a single twist conformation both in the solid-state and in solution in nonpolar solvents.

Abrahamsson, Bertil; Johansson, Dick; Torstensson, Arne; Wingstrand, Karin

doi: 10.1023/A:1018920412020pmid: 7971707

The drug release of felodipine, a water-insoluble drug, was tested by using sodium lauryl sulphate (SLS), polyoxyethylene 20 sorbitan monooleate (Tween) or cetyltrimethylammonium bromide (CTAB) in the test medium as solubilizers. Three slightly different felodipine extended-release (ER) tablets 10 mg based on the gel matrix principle were evaluated under different solubilizer concentrations, agitation intensities and pH. These tablets were also tested in a bioavailability study together with an oral solution. All three solubilizers substantially enhanced the drug solubility and sink conditions were obtained. The choice of solubilizer affected the drug release rate. This is most probably due to physico-chemical interactions between the gel-forming agent and the solubilizers. All in vitro test conditions provided a good correlation (r2 = 0.94 – 0.97) to in vivo dissolution, as determined by moment analysis. However, a much steeper in vitro/in vivo relationship was obtained for SLS compared to Tween and CTAB reflecting an inferior discrimination between the tablets by use of this anionic solubilizer.

Nail, Steven; Her, Lih-Min; Proffitt, Christopher; Nail, Lisa

doi: 10.1023/A:1018972428858pmid: 7971708

A microscope stage for observation of freezing and freeze drying is described. The stage uses thermoelectric (Peltier) heaters configured in two stages, with circulating fluid as a heat sink on the high temperature side. Lowest attainable sample temperature is about −47°C. Principal advantages of this system are closed-loop control of stage temperature, rapid response to changes in temperature set point, and improved documentation of experiments by use of a video recorder system with a character generator which allows display of sample identity and temperature. Accuracy of measuring the sample temperature in the field of view was validated by comparing observed values of eutectic melting with published values for a series of solutes with eutectic temperatures in the range from −2°C to −32°C. Good agreement was obtained throughout this range.

Niven, Ralph; Lott, Fred; Ip, Anna; Cribbs, Judith

doi: 10.1023/A:1018924512928pmid: 7971709

Two powder formulations (MMAD <4 µm) containing rhG-CSF were insufflated (IF) via an endotracheal tube at doses of 5, 75 or 500 µg/kg to New Zealand white rabbits. Doses of 5 and 500 µg/kg of solutions were administered by intratracheal instillation (IT), subcutaneous (SC) injection in the thigh and intravenous injection (IV) via the marginal ear vein. Blood samples were removed at regular intervals from an indwelling jugular catheter. Blood was analyzed directly for total white blood cell counts (WBC). Plasma was assayed for rhG-CSF by a specific ELISA. The distribution of radioactive dose in lung tissue was found after administering Tc99m HSA in solution or when incorporated into powders. The pharmacokinetics and pharmacodynamics were determined for all routes of administration. High dose IV concentration vs. time profiles declined biexponentially (t1/2 α = 0.6 ± 0.2 hrs, t1/2 β = 4.6 ± 0.2 hrs, n = 8). Clearance was dose dependent (11.6 ± 2.6 [500 µg/kg, n = 8] vs. 21.8 ± 3.3 ml/hr/kg [5 µg/kg, n = 5]). A normal systemic response was obtained after IF, indicating that rhG-CSF retains activity in the solid state. Dissolution and absorption of rhG-CSF from the powders were not rate limiting. The plasma concentration vs. time profiles peaked at similar times to those after IT (Tmax 1 -2 hrs) but were earlier than obtained after SC (Tmax 6-10 hrs). Powders were less efficiently dosed to the lung lobes after insufflation compared with instillates (14.7 ± 10.5 vs. 60.1 ± 10.6%), resulting in bioavailabilities ranging from 5 to 33%. Bioavailability after SC was 11.0 ± 7.0% and 95.3 ± 7.9% (n = 6) for the low and high doses, respectively.

Harrison, Jeannine; Shi, Xianglin; Wang, LiYing; Ma, Joseph; Rojanasakul, Yongyut

doi: 10.1023/A:1018976529766pmid: 7971710

Excessive production of reactive oxygen species by alveolar macrophages (AMs) in response to inhaled toxic substances is a major cause of oxidative lung injury. Therapeutic approaches designed to protect the lungs from oxidative injury by administering native antioxidant enzymes such as catalase and superoxide dismutase have been suggested. However, problems associated with poor penetration of these enzymes to the intracellular target sites have limited their effective use. The present study reports a drug targeting method based on receptor-mediated endocytosis of the antioxidant enzyme catalase to the AMs. This method employs molecular conjugate consisting of a cognate moiety, in this case IgG which recognizes the macrophage Fc receptor, covalently linked to the enzyme catalase via the reversible disulfide linkage. The uptake efficiency of the enzyme conjugate and its protection against oxidative injury were evaluated microfluorometrically using the intracellular oxidative probe dichlorodihydrofluorescein BSA: anti BSA antibody complex (DCHF-IC), and the cell viability indicator propidium iodide. The DCHF-IC-stimulated macrophages exhibited a dose- and time-dependent increase in intracellular fluorescence with a half maximal response dose of approximately 120 µg/ml. Free catalase (50–500 U/ml) failed to inhibit the DCHF-IC-induced oxidative burst and had only a marginal protective effect on AM injury. In contrast, the catalase-IgG conjugate (50–500 U/ml) strongly inhibited both the DCHF-IC-induced oxidation and injury in a dose-dependent manner. Effective inhibition was shown to require both the antioxidant catalase moiety and the cognate moiety for the cell surface receptor. Specific internalization of the conjugate through the Fc receptor was also investigated by competitive inhibition using free IgG. Under this condition, the conjugate showed a much reduced protective effect on intracellular oxidation, indicating conjugate internalization through the Fc endocytosis pathway. Thus, the enzyme-IgG conjugate system may be used as an effective and selective means to deliver antioxidant enzymes to the intracellular oxidative targets of the AMs.

Asada, Hiroyuki; Douen, Takayuki; Mizokoshi, Yoshiko; Fujita, Takuya; Murakami, Masahiro; Yamamoto, Akira; Muranishi, Shozo

doi: 10.1023/A:1018928613837pmid: 7971711

The stability of insulin and its acyl derivatives in the small intestine was examined in vitro. When these compounds were incubated in small intestinal fluid at 37°C, proteolysis of monoacyl insulins was reduced by increasing the carbon number of the fatty acid attached to Phe-B1 of the insulin molecule. In contrast, Phe-B1 and Lys-B29 diacylated insulins were more susceptible to hydrolysis than native insulin. Similar results were obtained using homogenates of the small intestinal mucosa, although the extent of the contribution of acylation to insulin degradation differed. The mechanism of the accelerated insulin proteolysis by diacylation was studied by circular dichroism (CD). The negative maxima at 270 nm in the CD spectra were attenuated for the diacyl derivatives, indicating that insulin association was inhibited by diacylation. Therefore, the increased proportion of monomers available for insulin proteolysis represents a main factor that makes diacyl derivatives unstable.

Deshpande, Deepa; Toledo-Velasquez, David; Wang, Li; Malanga, Carl; Ma, Joseph; Rojanasakul, Yongyut

doi: 10.1023/A:1018980630675pmid: 7971712

The present study investigated the feasibility of utilizing receptor-mediated endocytosis as a means to enhance peptide delivery to the pulmonary epithelium. The strategy employs a molecular conjugate consisting of a cognate moiety, transferrin (TF), covalently-linked to a model polypeptide, horseradish peroxidase (HRP), via a reversible disulfide linkage. A cultured alveolar epithelial monolayer system was used to simulate the conditions of the pulmonary epithelium and to allow accurate quantitation of intra- and transcellular peroxidase transport. The alveolar cells were isolated from rat lungs by enzymatic digestion and grown on microporous tissue culture-treated polycarbonate filters. A significant increase in the uptake of HRP by the cell monolayer was observed upon its conjugation with TF. The effect was found to be concentration-dependent, being more pronounced at low concentrations, i.e., 3.9- and 1.2-fold increase over unconjugated HRP controls at the concentration levels of 0.05 and 1.50 U/ml respectively. Effective peroxidase uptake was shown to require the TF cognate moiety for the cell surface receptor. Specific internalization of the conjugate by the TF endocytic pathway was verified by competition for the TF receptor. Conjugate internalization was not followed by a proportional increase in transcytosis, i.e., at 0.05 U/ml conjugate level, a 1.7-fold increase in transcytosis was observed as compared to 3.9-fold for endocytosis. Effective enhancement of transcytosis was achieved by treating the monolayers with brefeldin A (BFA), a compound known to affect intracellular transport of TF receptor complexes. At 1.6 µ/ml concentration level, BFA promoted a >20-fold increase in the rate of transcytosis of the conjugate in both the apical-to-basal and basal-to-apical directions. This effect was not associated with membrane leakage since BFA-treated monolayers maintained tight barrier to transport of the paracellular permeability solute 14C mannitol. In addition, BFA had no significant effect on the transport of free HRP. Instead, the effect of BFA on conjugate transport was mediated by TF receptors since excess free TF competitively inhibited transcytosis of the conjugate. Thus, our results are consistent with the TF receptor-mediated transport of the conjugate and its enhancement through the intracellular rerouting of the conjugate by BFA. The findings in this study may potentially be relevant to the design of drug delivery systems that can enhance intra- or transcellular uptake of therapeutic peptides in the pulmonary epithelium.