Antiherpes simplex virus type 2 activity of the antimicrobial peptide subtilosin.

AIMS: In this study, we evaluated the antiviral activity of subtilosin, a cyclical peptide isolated from Bacillus amyloliquefaciens, against herpes simplex virus type 2 (HSV-2) in cell cultures and we investigated subtilosin mode of action. METHODS AND RESULTS: We determined, using a virus yield inhibition assay, that noncytotoxic concentrations of subtilosin inhibit HSV-2 replication in Vero cell cultures. Subtilosin strongly inhibited extracellular and total virus production even when it was added at 8 h postinfection indicating that not only virus release but also viral particle formation is impeded by the antiviral peptide. Although viral glycoprotein gD level of expression is not affected by the bacteriocin, an altered pattern of gD intracellular localization was detected by immunofluorescence assay in subtilosin-treated culture. On the other hand, at high concentrations, subtilosin displays virucidal action. CONCLUSIONS: Subtilosin displays antiviral and virucidal actions against HSV-2. The target of subtilosin inhibitory effect would be late stages of the viral replicative cycle such as viral glycoprotein intracellular transport. SIGNIFICANCE AND IMPACT OF THE STUDY: Given its antimicrobial activity and its safety for human tissues, subtilosin could represent a valuable alternative to be considered in the development of new microbicide formulations.

Toxicodynamics of rigid polystyrene microparticles on pulmonary gas exchange in mice: implications for microemboli-based drug delivery systems.

The toxicodynamic relationship between the number and size of pulmonary microemboli resulting from uniformly sized, rigid polystyrene microparticles (MPs) administered intravenously and their potential effects on pulmonary gas exchange were investigated. CD-1 male mice (6-8 weeks) were intravenously administered 10, 25 and 45 µm diameter MPs. Oxygen hemoglobin saturation in the blood (SpO(2)) was measured non-invasively using a pulse oximeter while varying inhaled oxygen concentration (F(I)O(2)). The resulting data were fit to a physiologically based non-linear mathematical model that estimates 2 parameters: ventilation-perfusion ratio (V(A)/Q) and shunt (percentage of deoxygenated blood returning to systemic circulation). The number of MPs administered prior to a statistically significant reduction in normalized V(A)/Q was dependent on particle size. MP doses that resulted in a significant reduction in normalized V(A)/Q one day post-treatment were 4000, 40,000 and 550,000 MPs/g for 45, 25 and 10 µm MPs, respectively. The model estimated V(A)/Q and shunt returned to baseline levels 7 days post-treatment. Measuring SpO(2) alone was not sufficient to observe changes in gas exchange; however, when combined with model-derived V(A)/Q and shunt early reversible toxicity from pulmonary microemboli was detected suggesting that the model and physical measurements are both required for assessing toxicity. Moreover, it appears that the MP load required to alter gas exchange in a mouse prior to lethality is significantly higher than the anticipated required MP dose for effective drug delivery. Overall, the current results indicate that the microemboli-based approach for targeted pulmonary drug delivery is potentially safe and should be further explored.

Prodrug and conjugate drug delivery strategies for improving HIV/AIDS therapy.

Despite the wide variety of highly potent anti-HIV drugs that have been developed and made available in clinical practice over the years, eradication of HIV infection has not been achieved. Currently, HIV infection and AIDS are thought to be chronically treatable. HIV attacks host immune cells namely macrophages and CD4(+)T-cells and sequesters itself into sanctuary and reservoir sites such as the lymphoid tissues, testes, and brain. Initial drug delivery efforts with prodrugs and drug conjugates focused on improving the physicochemical (i.e. solubility), biopharmaceutic (i.e. absorption, metabolism), and pharmacokinetic (i.e. blood concentrations) properties of the parent drugs. Eradicating HIV, however, will require advanced drug delivery approaches in order to access and maintain effective drug concentrations for prolonged periods of time in sanctuary sites. The current review discusses prodrug/conjugate efforts, clinical successes and describes drug delivery challenges and approaches for eradicating HIV infection.

siRNA--getting the message out.

The recent observation that potent and sequence-specific gene silencing by injection of double-stranded RNA (dsRNA) has sparked the phenomenon known as "RNA interference" (RNAi) and has enabled the gene-specific knockdown of drug transport proteins and metabolizing enzymes. The application of small interfering RNAs (siRNAs) is broad and the potential for use as research tools is now well established in vitro. In vivo use is still a challenge that is primarily focused on the difficulty of delivering siRNAs to target cells. The potential use of siRNAs as therapeutic agents is also exciting and holds great promise for future. For the study of drug transporter function in absorption, distribution, metabolism and excretion (ADME) and in the treatment of diseases, siRNA offers a way to gather interpretable mechanistic data-a distinct advantage over the use of "specific" chemical inhibitors. This mini review provides background information on siRNA as well as examples of the use of siRNA as applied to drug transporters.

Tumor-specific targeting of an anticancer drug delivery system by LHRH peptide.

The central problem in cancer chemotherapy is the severe toxic side effects of anticancer drugs on healthy tissues. Invariably the side effects impose dose reduction, treatment delay, or discontinuance of therapy. To limit the adverse side effects of cancer chemotherapy on healthy organs, we proposed a drug delivery system (DDS) with specific targeting ligands for cancer cells. The proposed DDS minimizes the uptake of the drug by normal cells and enhances the influx and retention of the drug in cancer cells. This delivery system includes three main components: (i) an apoptosis-inducing agent (anticancer drug), (ii) a targeting moiety-penetration enhancer, and (iii) a carrier. We describe one of the variants of such a system, which utilizes camptothecin as an apoptosis-inducing agent and poly(ethylene glycol) as a carrier. Luteinizing hormone-releasing hormone (LHRH) was used as a targeting moiety (ligand) to LHRH receptors that are overexpressed in the plasma membrane of several types of cancer cells and are not expressed detectably in normal visceral organs. The results showed that the use of LHRH peptide as a targeting moiety in the anticancer DDS substantially enhanced the efficacy of chemotherapy, led to amplified apoptosis induction in the tumor, and minimized the side effects of the anticancer drug on healthy organs. The LHRH receptor targeting DDS did not show in vivo pituitary toxicity and did not significantly influence the time course or the plasma concentration of luteinizing hormone and its physiological effects on the reproductive functions of mice.

Targeted PEG-based bioconjugates enhance the cellular uptake and transport of a HIV-1 TAT nonapeptide.

We previously described the enhanced cell uptake and transport of R.I-K(biotin)-Tat9, a large ( approximately 1500 Da) peptidic inhibitor of HIV-1 Tat protein, via SMVT, the intestinal biotin transporter. The aim of the present study was to investigate the feasibility of targeting biotinylated PEG-based conjugates to SMVT in order to enhance cell uptake and transport of Tat9. The 29 kDa peptide-loaded bioconjugate (PEG:(R.I-Cys-K(biotin)-Tat9)8) used in these studies contained eight copies of R.I-K(biotin)-Tat9 appended to PEG by means of a cysteine linkage. The absorptive transport of biotin-PEG-3400 (0.6-100 microM) and the bioconjugate (0.1-30 microM) was studied using Caco-2 cell monolayers. Inhibition of biotin-PEG-3400 by positive controls (biotin, biocytin, and desthiobiotin) was also determined. Uptake of these two compounds was also determined in CHO cells transfected with human SMVT (CHO/hSMVT) and control cells (CHO/pSPORT) over the concentration ranges of 0.05-12.5 microM and 0.003-30 microM, respectively. Nonbiotinylated forms of these two compounds, PEG-3350 and PEG:(R.I-Cys-K-Tat9)8, were used in the control studies. Biotin-PEG-3400 transport was found to be concentration-dependent and saturable in Caco-2 cells (K(m)=6.61 microM) and CHO/hSMVT cells (K(m)=1.26 microM). Transport/uptake was significantly inhibited by positive control substrates of SMVT. PEG:(R.I-Cys-K(biotin)Tat9)8 also showed saturable transport kinetics in Caco-2 cells (K(m)=6.13 microM) and CHO/hSMVT cells (K(m)=8.19 microM). Maximal uptake in molar equivalents of R.I-Cys-K(biotin)Tat9 was 5.7 times greater using the conjugate versus the biotinylated peptide alone. Transport of the nonbiotinylated forms was significantly lower (P<0.001) in all cases. The present results demonstrate that biotin-PEG-3400 and PEG:(R.I-Cys-K(biotin)Tat9)8 interact with human SMVT to enhance the cellular uptake and transport of these larger molecules and that targeted bioconjugates may have potential for enhancing the cellular uptake and transport of small peptide therapeutic agents.

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 microM) and R.I.-K(biotin)-Tat9 (0.1-25 microM) 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 microM) was determined in CHO/hSMVT and CHO/pSPORT (control) cells. RESULTS: The absorptive transport of R.I.-K-Tat9 was passive, low (Pm approximately 1 x 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 (Ea = 9.1 kcal/mole), concentration dependent and saturable transport (Km = 3.3 microM). R.I.-K(biotin)-Tat9 uptake in CHO/hSMVT cells (Km = 1.0 microM) was - 500-fold greater than R.I.-K-Tat9 (at 10 microM). 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.

Pharmacokinetic studies in Tg.AC and FVB mice administered [14C] benzene either by oral gavage or intradermal injection.

Chronic benzene toxicity has been demonstrated to result in either aplastic anemia or acute myelogenous leukemia, a form of granulocytic leukemia, in exposed people (Snyder and Kalf, Crit. Rev. Toxicol. 24, 177-209, 1994). Aplastic anemia has been demonstrated in animal models following benzene exposure but, heretofore, it has not been possible to replicate benzene-induced granulocytic leukemia in animals. The Tg.AC mouse appears to be the first animal model in which a granulocytic leukemia was produced by treatment with benzene (Tennant et al., The Use of Short- and Medium-Term Tests for Carcinogenic Hazard Evaluation, 1999; French and Saulnier, J. Toxicol. Environ. Health 61, 377-379, 2000). Leukemia was observed in Tg.AC mice to which benzene was administered dermally. Neither orally dosed Tg.AC mice or mice of the parental FVB strain treated by either route of exposure developed leukemia. It is well established that benzene metabolism is required to produce benzene toxicity. To determine whether metabolic differences arising from differences in route of exposure or strain of mouse directed the development of leukemia, the pharmacokinetics of benzene were compared between the two strains and between the two routes of administration. Regardless of the route of exposure or the strain of mouse, seven major metabolites plus unmetabolized benzene were detected in most samples at most time points. Few differences were observed between the two strains following either route of administration. These results suggest that the genetic modification in the Tg.AC mouse, i.e., insertion of the v-Ha-ras construct into the genome, did not disrupt any major pathways involved in determining the pharmacokinetics of benzene. Two significant differences were observed between the two routes of exposure: first, benzene was absorbed more slowly after intradermal injection than after oral gavage, and second, the intradermally dosed mice produced more conjugates of hydroquinone than did the orally dosed mice. These differences in metabolism may be involved in the previously observed differences in hematotoxicity between the two routes of exposure.

Development of a novel intestinal and vascular access port (IVAP) rabbit model to study regiospecific oral absorption pharmacokinetics.

BACKGROUND AND PURPOSE: The limited availability and cost of many drugs prohibits routine use of the previously developed intestinal and vascular access port (IVAP) canine model by our group. A lower animal species model such as the rabbit is suitable for implanting intestinal and vascular access ports for investigating regiospecific intestinal absorption and hepatic elimination while requiring significantly lower doses of drugs. In addition, expression of certain cytochrome P450 enzymes and apical secretory and absorptive transporters in rabbit intestine is similar to that in humans making the rabbit a suitable model. METHODS: Individual 5-F Silastic catheters were placed in the proximal or distal portion of the small intestine or the colon of subject animals, while a 5-F Heparin Coated Polyurethane (HCP) catheter was implanted in the portal vein of each subject. The catheters were tunneled out of the abdomen and attached to separate subcutaneous access ports along the spine. The animals were allowed a two-week minimum recovery period prior to being used in pharmacokinetic studies. RESULTS AND DISCUSSION: After some initial difficulties, rabbits with IVAP implants proved to be an efficient and dependable model for investigating intestinal and hepatic extraction of drugs. Fluoroscopic visualization of intestinal and portal venous catheters indicated that surgically implanted catheters did not interfere with gastrointestinal motility or blood flow into the liver, respectively. Acute pH studies in the proximal portion of the small intestine were consistent with normal GI motility patterns.

Differentiation of gut and hepatic first-pass effect of drugs: 1. Studies of verapamil in ported dogs.

PURPOSE: To investigate the relative contributions of the gut and liver to the first-pass loss of verapamil (VL) using an in vivo intestinal-vascular access port (IVAP) dog model. METHODS: Basic pharmacokinetics of VL were determined after intravenous (IV: 0.5 mg/kg), portal venous (PV: 2 mg/kg), and duodenal (ID: 2 mg/kg) administration in IVAP dogs. Serial blood samples were collected for 8 h after dosing, and plasma was analyzed for unchanged drug by a high-performance liquid chromatography-fluorescence method. Extraction ratios in the liver and intestinal tract were determined from the area under the concentration-time curves for ID, PV, and IV administration. The functional role of CYP450 or secretory transporters such as P-gp on the gut and liver first-pass loss of VL was further studied using ritonavir, a known substrate or inhibitor of these processes. RESULTS: The liver had a high intrinsic capacity for clearing VL because the absolute bioavailability (BA) of VL was 21.7% after PV administration. The BA of VL after ID administration was 23.5%; therefore, intestinal absorption was complete and intestinal extraction was negligible (ER(GI) approximately 0). The BA of VL increased from 23.5% to 66.2% in the presence of ritonavir primarily due to a reduction in hepatic extraction. CONCLUSIONS: Although the liver had a high intrinsic capacity for extracting VL, the contribution of gut to the first-pass loss of VL was negligible. Because of the additive effects of intestinal CYP3A-mediated metabolism and secretory transport, a significant gut first-pass effect was expected, but not observed in dogs. These studies demonstrate the utility of the in vivo IVAP dog model for evaluating the relative contribution of the gut and liver to the first-pass loss of drugs and for characterizing the functional role that CYP450 metabolism and/or secretory transporters play in drug-drug interactions and reduced oral bioavailability.

Active efflux kinetics of etoposide from rabbit small intestine and colon.

The aim of the present study was to investigate the directional transport kinetics of etoposide in rabbit intestinal tissues using side-by-side diffusion chambers. Etoposide is a routinely used mixed-mechanism 'efflux' inhibitor; however, its absorptive and secretory transport kinetics in rabbit intestinal tissues, a commonly used animal model, have not yet been reported. Kinetic studies revealed that the apical (AP) to basolateral (BL) (i.e. absorptive) transport of etoposide was not apparently mediated by specialized transporters, whereas secretion (i.e. BL to AP transport) by intestinal tissues was concentration dependent and saturable. Half-saturation constant values (K(m), mean+/-standard deviation (S.D.)) ranged from 53.6+/-35.8 microM to 168.7+/-127.3 microM, consistent with previous results from our group in intestinal tissues from other species and Caco-2 cell monolayers. Secretory permeability was greatest in the ileum, whereas values in the upper small intestine and colon were approximately equal, and represented only 50% of the value in the ileum. The ileal secretory transport of etoposide was temperature dependent, with the activation energy (E(a)) >4 kCal/mole at 5 microM, suggesting the involvement of the active, energy dependent mechanism. Etoposide inhibition by verapamil and saquinavir, known inhibitors of intestinal secretion, was characterized as competitive with K(i)'s equal to 193.0+/-164.4 microM and 72.6+/-53.5 microM, respectively. The current results demonstrate that the absorptive transport of etoposide in rabbit tissue was not mediated by specialized carriers, and that secretory transport was regionally dependent, mediated by a transporter or transporters, the K(m)'s were in the micromolar range, and involved the energy dependent mechanism(s). The relatively low k(m) of etoposide compared with its aqueous solubility (0.25-0.34 mM, pH 5-6.5, 25 degrees C) makes it the excellent mixed-mechanism competitive inhibitor for determining the secretory transport properties of putative drug substrates. Understanding the in vitro secretory transport kinetics of etoposide provides a mechanistic basis for ongoing studies exploring the functional role of 'efflux' in vivo.

Adjuvancy enhancement of muramyl dipeptide by modulating its release from a physicochemically modified matrix of ovalbumin microspheres. I. In vitro characterization.

The weak immunogenicity of subunit vaccines has necessitated research into the development of novel adjuvants and methods to enhance the adjuvancy associated with vaccine delivery systems. The purpose of the present study was to modulate the release of muramyl dipeptide (MDP) from a physicochemically modified matrix of ovalbumin microspheres (OVA-MSs). A two-component MS vaccine delivery system was fabricated, which utilized OVA as the antigen and delivery matrix, and MDP as the adjuvant. The MSs were prepared from OVA using a water/oil emulsion method, followed by suspension cross-linking using glutaraldehyde. The MS matrix was modified with respect to the degree of cross-linking by varying the concentration of glutaraldehyde and matrix density, a function of disulfide-bond formation. The modifications in the MS matrix were characterized using SDS-PAGE, scanning electron microscopy, differential scanning calorimetry, and thin layer wicking (TLW). The in vitro release of MDP and OVA from the various preparations of OVA-MSs exhibited triphasic and biphasic profiles, respectively. The degree of cross-linking and the matrix density were found to be significant physicochemical parameters that affected the release profiles of MDP and OVA through two mechanisms: controlled surface erosion and bulk degradation of the MSs.

Adjuvancy enhancement of muramyl dipeptide by modulating its release from a physicochemically modified matrix of ovalbumin microspheres. II. In vivo investigation.

In the present study, sustaining the release of adjuvants was investigated using microspheres as a means to increase the immune response (i.e. efficacy) and, ultimately, to reduce adverse effects to vaccine components. To date, most attempts have focused on sustaining the release of antigens. The utility of currently used vaccine adjuvants may be improved by sustaining their release. The development, modification and characterization of a two-component microsphere vaccine delivery system was demonstrated in our previous report [Puri et al., J. Control. Release (2000) in press]. Briefly, ovalbumin (OVA) was utilized as the model antigen (Ag) and delivery matrix and MDP or threonyl-MDP served as the model adjuvants. The release pattern of MDP was modulated from a physicochemically modified matrix of OVA microspheres (OVA-MSs). The purpose of the present study was to evaluate the adjuvancy of MDP in mice by modulating its release from OVA-MSs. Mice were immunized intradermally (i.d.) with various preparations of OVA-MSs, using a single-shot-immunization technique. Positive and negative control preparations were evaluated as well. An inverse relationship was observed between the in vitro release rate of MDP and the in vivo OVA-specific IgG antibody (Ab) immune response in mice. These results demonstrated that modulating the release pattern of MDP or threonyl-MDP enhanced their adjuvant effect. In conclusion, the current results demonstrate that the sustained and controlled release of adjuvants is extremely important for inducing a high level and prolonged period of immunostimulation while potentially minimizing therapy-limiting adverse effects.

Regional oral absorption, hepatic first-pass effect, and non-linear disposition of salmon calcitonin in beagle dogs.

The dose-dependent disposition, first pass hepatic elimination, and absorption pharmacokinetics (PK) of salmon calcitonin (sCT) were investigated in a canine Intestinal Vascular Access Port (IVAP) model. The PK of sCT were determined after intravenous (IV), subcutaneous (SC), portal venous (PV), and oral (PO) administration of sCT. Regional oral absorption of unformulated sCT was also evaluated by direct administration into the duodenum (ID), ileum (IL), and colon (IC) by means of surgically implanted, chronic catheters. Plasma samples were collected and analyzed by radioimmunoassay (RIA). Salmon calcitonin PK were evaluated using 2-compartmental and model independent methods. Intravenous sCT PK were non-linear over the dose range studied. High dose groups (100-1000 microg) demonstrated higher total plasma clearance (CL) and V(dss) than the low dose groups (1-25 microg). However, the MRT did not change for doses ranging from 10 to 1000 microg. After SC administration, the absorption of sCT was rapid with bioavailability (BA) varying from 21.4 to 52.9%. However, the BA of sCT was low after ID, IL, and IC administration (0.039, 0.064, and 0.021%, respectively). The role of hepatic first-pass elimination was negligible. The results of these studies demonstrate that the elimination of sCT is rapid but does not occur in the liver. Enhanced sCT clearance at higher doses was indicated by increasing V(dss) values, and it is hypothesized that increased renal blood flow and/or saturated plasma protein binding may contribute to the non-linear behavior. The IVAP canine model was found to have utility for probing the absorption and disposition PK of sCT. The combination of high oral bioavailability variability and non-linear disposition of sCT may produce highly variable therapeutic effects. The practical impact of the non-linear disposition of sCT remains to be determined. Based on the current results it appears that the rate-limiting step to the successful oral administration of sCT is its delivery into the portal vein since hepatic metabolism was negligible.

Oral delivery of salmon calcitonin.

Calcitonin plays a crucial role in both calcium homeostasis and bone remodeling. Establishing an oral delivery system for CT is of great importance since CT is currently administered only parenterally or nasally. Poor absorption and rapid proteolytic degradation have impeded the clinical development of an orally administered sCT drug product. Potential approaches to enhance sCT absorption include the use of formulation additives in the drug product to transiently modulate the intestinal environment or targeting specific intestinal regions that may have favorable peptide delivery properties (e.g., low residual volume, high absorptive surface area or reduced enzymatic activity). Potential approaches to limit the activity of intestinal enzymes include adjusting the pH of the intestinal contents to the pH minima of specific enzymes or maintaining high local drug concentrations in order to saturate enzyme systems. In this review, pharmacokinetic studies elucidating the rate-limiting steps for achieving adequate sCT oral bioavailability are detailed. Further, several approaches for enhancing the oral absorption of sCT are presented. Specific emphasis is placed on regio-specific targeting (e.g., intestinal regional differences in dilution and spreading, etc.) and modulation of the intestinal environment (e.g., changing pH, etc.). The approaches are evaluated in in vitro and in vivo models. Finally, this paper closes with a brief section of concluding remarks.

Improved longevity and functionality of a canine model providing portal vein and multi-site intestinal access.

BACKGROUND AND PURPOSE: The canine intestinal and venous access port (IVAP) model is valuable for investigating hepatic elimination and region-specific intestinal absorption of pharmaceuticals. Previously, long-term functionality of this preparation has been variable. METHODS: Catheters of different construction were placed in the proximal and distal portions of the small intestine, colon, and portal vein of subject animals and were attached to separate subcutaneous access ports. Intraoperative, postoperative, and long-term maintenance techniques were developed, modified, and analyzed. RESULTS: Intestinal catheter infections and access site failures were associated with breakdown at the intestinal insertion site. The ileal catheter was prone to obstruction with ingesta. A modified Witzel technique, specialized port-catheter systems, scheduled port-flushing methods, and venous port infection treatment protocols improved the model's longevity. CONCLUSIONS: The canine IVAP model is a powerful tool for investigation of regional differences in intestinal absorption and hepatic elimination of drugs. Other researchers can derive increased longevity with the IVAP model by using the technical modifications detailed here: strict sterile technique, closed-end slit-valve catheters, GPV ports, the Witzel tunnel technique, routine portal vein infection surveillance, 50% dextrose intestinal catheter infusion, rapid removal of infected intestinal catheters, and critical appraisal of their results. Longevity of the model continues to be improved.

Regional differences in intestinal spreading and pH recovery and the impact on salmon calcitonin absorption in dogs.

PURPOSE: To investigate the regional influence of intestinal spreading and pH recovery on the performance of drug and excipient delivery systems and their impact on the oral absorption of a model peptide drug, salmon calcitonin (sCT), in conscious beagle dogs. METHODS: Male beagle dogs were surgically prepared with subdermal Intestinal Access Ports (IAP). The catheter from one port was placed in the duodenum and the other in the ileum. Fluoroscopy and Heidelberg pH capsule studies were performed to characterize intestinal spreading and pH recovery, respectively. Three treatments were performed: (1) a radiopaque dye and citric acid (CA) were infused into the intestinal segments, (2) a radiopaque powder capsule containing CA was given orally, and (3) capsules containing CA and sCT were given orally. Regular blood samples were collected and analyzed by radioimmunoassay (RIA) to determine the absorption characteristics of sCT. RESULTS: Since sCT is an excellent substrate for the pancreatic serine protease trypsin, the rate of degradation of sCT in the GI lumen is dependent upon the regional pH, activity of digestive enzymes and the concentration of sCT at the site of absorption. Fluoroscopy results clearly showed that when the radiopaque dye was infused into the duodenum and capsule disintegration occurred early, there was significant dilution and spreading of the excipients throughout a large section of the upper small intestine (USI). However, when the radiopaque dye was infused into the ileum and capsule disintegration occurred in the lower small intestine (LSI), the excipients moved along as a bolus (i.e., plug). The pH monitoring results were consistent with the fluoroscopy results. The pH dropped only momentarily and rose quickly in the USI consistent with well-stirred mixing kinetics. In the LSI, dilution and spreading were minimal and the drop in pH was greater and persisted for a longer period of time. Plasma levels of sCT were maximal when disintegration occurred in the LSI. CONCLUSIONS: Since significantly less dilution and spreading occurred in the LSI, the exposure of the intestine to pharmaceutical excipients and sCT was more concentrated resulting in a higher fraction of sCT absorbed. The results of this study demonstrate that intestinal mixing kinetics have a dramatic impact on the ability of pharmaceutical excipients to modulate the oral bioavailability of peptide drugs like sCT.

An investigation of the intradermal route as an effective means of immunization for microparticulate vaccine delivery systems.

Among the common routes of parenteral immunization, the skin is the only site that can function as an immune organ. Skin-associated lymphoid tissue contains specialized cells that enhance the immune response. The intercellular space in the skin interstitium provides a connection to the lymphatic capillaries and vessels that terminate in peripheral immune organs like the lymph nodes and spleen. The potential of intradermal immunization with microparticulate vaccine delivery systems was investigated in this study. The microparticulates used were muramyl dipeptide (MDP) loaded ovalbumin microspheres (OVA-MSs) and fluorescent latex microspheres of fixed sizes of 2.3 and 2.1 microm diameter, respectively. Similar doses of OVA-MSs were injected subcutaneously (s.c.) and intradermally (i.d.) in mice. The induced OVA-specific IgG antibody immune response was found to be significantly higher in i.d. immunized mice as compared to those injected s.c. The sc and i.d. administration of fluorescent latex microspheres in mice demonstrated that the uptake and translocation of microspheres from the site of injection depends primarily upon the surface area of the microspheres. The enhancement in antibody production upon i.d. administration was explained on the basis of (i) an increased surface area of microspheres and a lower number of microspheres per injection site, and (ii) an increased probability of interaction with the immune cells of the skin. Efficient lymph node targeting observed from the id administered microspheres may be the result of both of these factors. The results of this study demonstrated that the intradermal route is an effective means of immunization for microparticulate vaccine delivery systems, requiring lower doses and resulting in a higher immune response as compared to the traditionally used sc route.

Development of predictive pharmacokinetic simulation models for drug discovery.

As discovery chemistry produces increased numbers of potential drug compounds, the use of ADME (absorption, distribution, metabolism, and excretion) properties is becoming increasingly important in the drug selection and promotion process. A computer simulation model has been developed and validated to predict ADME outcomes, such as rate of absorption, extent of absorption, etc. using a limited number of in vitro data inputs. The oral bioavailability of ganciclovir in dogs and humans was simulated using a physiologically based model that utilized many biopharmaceutically relevant parameters, such as the concentration of ganciclovir in the duodenum, jejunum, ileum, and colon at various dose levels and solubility values. The simulations were run and compared to dog and human in vivo data. The simulation results demonstrated that the low bioavailability of ganciclovir is limited by compound solubility rather than permeability due to partitioning as previously speculated. This technology provides a breakthrough in in silico prediction of absorption and with its continued development and improvement, will aid drug discovery and development scientists to produce better pharmaceutical products.