Silk-elastinlike copolymers enhance bioaccumulation of semisynthetic glycosaminoglycan ethers for prevention of radiation induced proctitis.

Radiation-induced proctitis (RIP) is a debilitating adverse event that occurs commonly during lower abdominal radiotherapy. The lack of prophylactic treatment strategies leads to diminished patient quality of life, disruption of radiotherapy schedules, and limitation of radiotherapy efficacy due to dose-limiting toxicities. Semisynthetic glycosaminoglycan ethers (SAGE) demonstrate protective effects from RIP. However, low residence time in the rectal tissue limits their utility. We investigated controlled delivery of GM-0111, a SAGE analogue with demonstrated efficacy against RIP, using a series of temperature-responsive polymers to compare how distinct phase change behaviors, mechanical properties and release kinetics influence rectal bioaccumulation. Poly(lactic acid)-co-(glycolic acid)-block-poly(ethylene glycol)-block-poly(lactic acid)-co-(glycolic acid) copolymers underwent macroscopic phase separation, expelling >50% of drug during gelation. Poloxamer compositions released GM-0111 cargo within 1 h, while silk-elastinlike copolymers (SELPs) enabled controlled release over a period of 12 h. Bioaccumulation was evaluated using fluorescence imaging and confocal microscopy. SELP-415K, a SELP analogue with 4 silk units, 15 elastin units, and one elastin unit with lysine residues in the monomer repeats, resulted in the highest rectal bioaccumulation. SELP-415K GM-0111 compositions were then used to provide localized protection from radiation induced tissue damage in a murine model of RIP. Rectal delivery of SAGE using SELP-415K significantly reduced behavioral pain responses, and reduced animal mass loss compared to irradiated controls or treatment with traditional delivery approaches. Histological scoring showed RIP injury was ameliorated for animals treated with GM-0111 delivered by SELP-415K. The enhanced bioaccumulation provided by thermoresponsive SELPs via a liquid to semisolid transition improved rectal delivery of GM-0111 to mice and radioprotection in a RIP model.

A Phase 2b randomized trial of lorecivivint, a novel intra-articular CLK2/DYRK1A inhibitor and Wnt pathway modulator for knee osteoarthritis.

OBJECTIVE: Lorecivivint (LOR; SM04690), an investigational Wnt pathway modulator, previously demonstrated patient-reported and radiographic outcome improvements vs placebo in clinically relevant subjects with moderate to severe knee osteoarthritis (OA). This study's objective was to identify effective LOR doses. DESIGN: Subjects in this 24-week, Phase 2b, multicenter, randomized, double-blind, placebo (PBO)-controlled trial received an intra-articular injection of 2 mL LOR (0.03, 0.07, 0.15, or 0.23 mg), PBO, or dry-needle sham. The primary efficacy endpoints were changes in Pain NRS [0-10], WOMAC Pain [0-100], WOMAC Function [0-100], and radiographic mJSW outcomes, which were measured using baseline-adjusted analysis of covariance at Week 24. Multiple Comparison Procedure-Modeling (MCP-Mod) was performed for dose modeling. RESULTS: In total, 695/700 subjects were treated. Pain NRS showed significant improvements vs PBO after treatment with 0.07 mg and 0.23 mg LOR at Weeks 12 (-0.96, 95% CI [-1.54, -0.37], P = 0.001; -0.78 [-1.39, -0.17], P = 0.012) and 24 (-0.70 [-1.34, -0.06], P = 0.031; -0.82 [-1.51, -0.12], P = 0.022). Additionally, 0.07 mg LOR significantly improved WOMAC Pain and Function subscores vs PBO at Week 12 (P = 0.04, P = 0.021), and 0.23 mg LOR significantly improved both WOMAC subscores at Week 24 (P = 0.031, P = 0.017). No significant differences from PBO were observed for other doses. No radiographic progression was observed in any group at Week 24. MCP-Mod identified 0.07 mg LOR as the lowest effective dose. CONCLUSION: This 24-week Phase 2b trial demonstrated the efficacy of LOR on PROs in knee OA subjects. The optimal dose for future studies was identified as 0.07 mg LOR.

Severity of symptoms, physical functioning and satisfaction in patients with lumbar spinal stenosis: a validation study of the Iranian version of the Swiss Spinal Stenosis Score.

AIM: A common cause of low back pain is lumbar spinal stenosis (LSS). The Swiss Spinal Stenosis Score (SSS) is a well-known questionnaire that measures the severity of symptoms, physical functioning and patient's satisfaction in lumbar spinal stenosis. This study aimed to translate and validate the SSS in Iran. METHODS: A prospective clinical validation study was performed. Forward-backward procedure was applied to translate the original questionnaires into Persian. A sample of patients with lumbar spinal stenosis completed the questionnaire twice: at pre- and postoperative (6 months follow-up) assessments. To test reliability the internal consistency was assessed by the Cronbach's alpha coefficient. Validity was evaluated using the known groups comparison. In addition the Oswestry Disability Index was used to perform convergent validity. RESULTS: In all 121 patients were entered into the study. The mean age of patients was 62.3 (SD=10.2) years. The Cronbach's alpha coefficient for the SSS was 0.88. Validity was performed by known groups analysis and showed satisfactory results. The instrument discriminated well between the subgroups of patients who differed in age, severity of lumbar spinal stenosis, and the Self-Paced Walking Test (SPWT). The change in the Oswestry Disability Index strongly correlated with the change in patients' scores on the SSS; lending support to its good convergent validity (r=0.82; P<0.001). CONCLUSION: The Iranian version of Swiss Spinal Stenosis Score performed well and the findings suggest that it is a valid measure of symptoms, physical functioning and satisfaction among patients with lumbar spinal stenosis.

Efficacy and safety of a recombinant factor IX (Bax326) in previously treated patients with severe or moderately severe haemophilia B undergoing surgical or other invasive procedures: a prospective, open-label, uncontrolled, multicentre, phase III study.

Haemostatic management of haemophilia B patients undergoing surgery is critical to patient safety. The aim of this ongoing prospective trial was to investigate the haemostatic efficacy and safety of a recombinant factor IX (rFIX) (Bax326) in previously treated subjects (12-65 years, without history of FIX inhibitors) with severe or moderately severe haemophilia B, undergoing surgical, dental or other invasive procedures. Haemostatic efficacy was assessed according to a predefined scale. Blood loss was compared to the average and maximum blood loss predicted preoperatively. Haemostatic FIX levels were achieved peri- and postoperatively in 100% of subjects (n = 14). Haemostasis was 'excellent' intraoperatively in all patients and postoperatively in those without a drain, and 'excellent' or 'good' at the time of drain removal and day of discharge in those with a drain employed. Following the initial dose, the mean FIX activity level rose from 6.55% to 107.58% for major surgeries and from 3.60% to 81.4% for minor surgeries. Actual vs. predicted blood loss matched predicted intraoperative blood loss but was equal to or higher than (but less than 150%) the maximum predicted postoperative blood loss reflecting the severity of procedure and FIX requirements. There were no related adverse events, severe allergic reactions or thrombotic events. There was no evidence that BAX326 increased the risk of inhibitor or binding antibody development to FIX. BAX326 was safe and effective for peri-operative management of 14 subjects with severe and moderately severe haemophilia B.

Poly(amido amine) dendrimers as absorption enhancers for oral delivery of camptothecin.

Oral delivery of camptothecin has a treatment advantage but is limited by low bioavailability and gastrointestinal toxicity. Poly(amido amine) or PAMAM dendrimers have shown promise as intestinal penetration enhancers, drug solubilizers and drug carriers for oral delivery in vitro and in situ. There have been very limited studies in vivo to evaluate PAMAM dendrimers for oral drug delivery. In this study, camptothecin (5 mg/kg) was formulated and co-delivered with cationic, amine-terminated PAMAM dendrimer generation 4.0 (G4.0) (100 and 300 mg/kg) and anionic, carboxylate-terminated PAMAM generation 3.5 (G3.5) (300 and 1000 mg/kg) in CD-1 mice. Camptothecin associated to a higher extent with G4.0 than G3.5 in the formulation, attributed to an electrostatic interaction on the surface of G4.0. Both PAMAM G4.0 and G3.5 increased camptothecin solubilization in simulated gastric fluid and caused a 2-3 fold increase in oral absorption of camptothecin when delivered at 2 h. PAMAM G4.0 and G3.5 did not increase mannitol transport suggesting that the oral absorption of camptothecin was not due to tight junction modulation. Histologic observations of the epithelial layer of small intestinal segments of the gastrointestinal tract (GIT) at 4 h post dosing supported no evidence of toxicity at the evaluated doses of PAMAM dendrimers. This study demonstrates that both cationic (G.4) and anionic (G3.5) PAMAM dendrimers were effective in enhancing the oral absorption of camptothecin. Results suggest that drug inclusion in PAMAM interior controlled solubilization in simulated gastric and intestinal fluids, and increased oral bioavailability.

COMPARATIVE PHARMACOKINETICS OF PAMAM-OH DENDRIMERS AND HPMA COPOLYMERS IN OVARIAN-TUMOR-BEARING MICE.

The purpose of this study was to model data from a head to head comparison of the in vivo fate of hyper-branched PAMAM dendrimers with linear HPMA copolymers in order to understand the influence of molecular weight (MW), hydrodynamic size (Rh) and polymer architecture on biodistribution in tumor-bearing mice using compartmental pharmacokinetic analysis. Plasma concentration data was modeled by two-compartment analysis using Winnonlin® to obtain elimination clearance (E.CL) and plasma exposure (AUCplasma). Renal clearance (CLR) was calculated from urine data collected over 1 week. A plasma-tumor link model was fitted to experimental plasma and tumor data by varying the tumor extravasation (K4, K6) and elimination (K5) rate constants using multivariable constrained optimization solver in Matlab®. Tumor exposures (AUCtumor) were computed from area under the tumor concentration time profile curve by the linear trapezoidal method. Along with MW and Rh, polymer architecture was critical in affecting the blood and tumor pharmacokinetics of the PAMAM-OH dendrimers and HPMA copolymers. Elimination clearance decreased more rapidly with increase in hydrodynamic size for PAMAM-OH dendrimers as compared to HPMA copolymers. HPMA copolymers were eliminated renally to a higher extent than PAMAM-OH dendrimers. These results are suggestive of a difference in extravasation of polymers of varying architecture through the glomerular basement membrane. While the linear HPMA copolymers can potentially reptate through a pore smaller in size than their hydrodynamic radii in a random coil conformation, PAMAM dendrimers have to deform in order to permeate across the pores. With increase in molecular weight or generation, the deforming capacity of PAMAM-OH dendrimers is known to decrease, making it harder for higher generation PAMAM-OH dendrimers to sieve through the glomerulus as compared to HPMA copolymers of comparable molecular weights. PAMAM-OH dendrimer had greater tumor extravsation rate constants and higher tumor to plasma exposure ratios than HPMA copolymers of comparable molecular weights which indicated that in the size range studied, when in circulation, PAMAM-OH dendrimers had a higher affinity to accumulate in the tumor than the HPMA copolymers.

Transepithelial transport and toxicity of PAMAM dendrimers: implications for oral drug delivery.

This article summarizes efforts to evaluate poly(amido amine) (PAMAM) dendrimers as carriers for oral drug delivery. Specifically, the effect of PAMAM generation, surface charge and surface modification on toxicity, cellular uptake and transepithelial transport is discussed. Studies on Caco-2 monolayers, as models of intestinal epithelial barrier, show that by engineering surface chemistry of PAMAM dendrimers, it is possible to minimize toxicity while maximizing transepithelial transport. It has been demonstrated that PAMAM dendrimers are transported by a combination of paracellular and transcellular routes. Depending on surface chemistry, PAMAM dendrimers can open the tight junctions of epithelial barriers. This tight junction opening is in part mediated by internalization of the dendrimers. Transcellular transport of PAMAM dendrimers is mediated by a variety of endocytic mechanisms. Attachment or complexation of cytotoxic agents to PAMAM dendrimers enhances the transport of such drugs across epithelial barriers. A remaining challenge is the design and development of linker chemistries that are stable in the gastrointestinal tract (GIT) and the blood stream, but amenable to cleavage at the target site of action. Recent efforts have focused on the use of PAMAM dendrimers as penetration enhancers. Detailed in vivo oral bioavailability of PAMAM dendrimer-drug conjugates, as a function of physicochemical properties will further need to be assessed.

Comparative biodistribution of PAMAM dendrimers and HPMA copolymers in ovarian-tumor-bearing mice.

The biodistribution profile of a series of linear N-(2-hydroxylpropyl)methacrylamide (HPMA) copolymers was compared with that of branched poly(amido amine) dendrimers containing surface hydroxyl groups (PAMAM-OH) in orthotopic ovarian-tumor-bearing mice. Below an average molecular weight (MW) of 29 kDa, the HPMA copolymers were smaller than the PAMAM-OH dendrimers of comparable molecular weight. In addition to molecular weight, hydrodynamic size and polymer architecture affected the biodistribution of these constructs. Biodistribution studies were performed by dosing mice with (125)iodine-labeled polymers and collecting all major organ systems, carcass, and excreta at defined time points. Radiolabeled polymers were detected in organ systems by measuring gamma emission of the (125)iodine radiolabel. The hyperbranched PAMAM dendrimer, hydroxyl-terminated, generation 5 (G5.0-OH), was retained in the kidney over 1 week, whereas the linear HPMA copolymer of comparable molecular weight was excreted into the urine and did not show persistent renal accumulation. PAMAM dendrimer, hydroxyl-terminated, generation 6.0 (G6.0-OH), was taken up by the liver to a higher extent, whereas the HPMA copolymer of comparable molecular weight was observed to have a plasma exposure three times that of this dendrimer. Tumor accumulation and plasma exposure were correlated with the hydrodynamic sizes of the polymers. PAMAM dendrimer, hydroxyl-terminated, generation 7.0 (G7.0-OH), showed extended plasma circulation, enhanced tumor accumulation, and prolonged retention with the highest tumor/blood ratio for the polymers under study. Head-to-head comparative study of HPMA copolymers and PAMAM dendrimers can guide the rational design and development of carriers based on these systems for the delivery of bioactive and imaging agents.

Geometry and surface characteristics of gold nanoparticles influence their biodistribution and uptake by macrophages.

Spherical and rod-shaped gold nanoparticles with surface poly(ethylene glycol) (PEG) chains were characterized for size, shape, charge, poly dispersity and surface plasmon resonance. The nanoparticles were injected intravenously to 6-8-week-old female nu/nu mice bearing orthotopic ovarian tumors, and their biodistribution in vital organs was compared. Gold nanorods were taken up to a lesser extent by the liver, had longer circulation time in the blood, and higher accumulation in the tumors, compared with their spherical counterparts. The cellular uptake of PEGylated gold nanoparticles by a murine macrophage-like cell line as a function of geometry was examined. Compared to nanospheres, PEGylated gold nanorods were taken up to a lesser extent by macrophages. These studies point to the importance of gold nanoparticle geometry and surface properties on transport across biological barriers.

Abdominal obesity and the spectrum of global cardiometabolic risks in US adults.

OBJECTIVE: To compare the association of obesity and abdominal obesity with cardiometabolic risk factor burden and global estimated coronary heart disease (CHD) risk among multiethnic US adults. DESIGN: Cross-sectional, survey study. SUBJECTS: A total of 4456 participants (representing 194.9 million adults) aged 20-79 years in the 2003-2004 National Health and Nutrition Examination Survey (NHANES). MEASUREMENTS: Body mass index (BMI) and waist circumference (WC) measures, CHD risk factors and a 10-year estimated CHD risk based on Framingham algorithms. Obesity was defined as a BMI >or=30 kg/m(2) and abdominal obesity as a WC >88 cm in women and >102 cm in men. High CHD risk status included diabetes, cardiovascular disease (CVD) or a 10-year Framingham risk score of >20%. RESULTS: Overall, abdominal obesity was present in 42.3% of men and 62.5% of women and in 53.6% of whites, 56.9% of blacks and 50.5% of Hispanics (P<0.001 between gender and ethnicity). However, using International Diabetes Federation (IDF)-recommended WC cut points for Hispanics, the prevalence of abdominal obesity was 78.3%. Mean levels of low-density lipoprotein cholesterol (LDL-C), systolic and diastolic blood pressure, fasting glucose and C-reactive protein increased, and high-density lipoprotein cholesterol (HDL-C) decreased (P<0.001) according to BMI and WC categories, although these associations were attenuated in blacks for blood pressure, LDL-C, HDL-C and triglycerides. Of those with high WC, 25-35% had >or=3 cardiometabolic risk factors. High CHD risk among those with high WC was most common in men (27.9%) and non-Hispanic whites (23.9%). Persons with a high vs normal WC, adjusted for age, gender, ethnicity and BMI were more likely to have >or=3 cardiometabolic risk factors (odds ratio (OR)=5.1, 95% confidence interval (CI)=3.9-6.6) and were classified as high CHD risk (OR=1.5, 95% CI=1.1-2.0). CONCLUSION: The association of abdominal obesity with risk factors varies by ethnicity and is independently associated with high CHD risk status, further validating its clinical significance.

Adenoviral gene delivery to solid tumors by recombinant silk-elastinlike protein polymers.

PURPOSE: The purpose of this study was to investigate the potential of silk-elastinlike protein polymers (SELPs) in controlling the release rate of adenoviruses in vitro and in vivo while preserving their bioactivity. MATERIALS AND METHODS: A hydrogel system composed of SELP/adenovirus mixture was prepared. The release of the adenovirus particles from the hydrogels was quantified by Real Time-PCR and the bioactivity of the released viruses was evaluated using confocal microscopy and beta-galactosidase assay. To demonstrate the ability of SELP in entrapping virus cargo and releasing it over a prolonged period of time in vivo, a SELP/adenovirus mixture was prepared and injected directly into xenograft tumor models of breast and head and neck cancer in mice. At various time points mice were sacrificed, tumors dissected, and tissue sections studied under confocal microscope. RESULTS: In vitro studies demonstrated that SELP hydrogels release viruses over a period of 4 weeks while preserving their bioactivity. After intratumoral injection, a prolonged and localized expression of adenoviruses was observed. CONCLUSIONS: These results suggest the potential of SELPs in local adenoviral delivery to solid tumors as an alternative approach to intratumoral virus infusion.

Recombinant polymer-protein fusion: a promising approach towards efficient and targeted gene delivery.

BACKGROUND: Synthetic vectors such as polymers have the potential to reduce the safety problems associated with viral vectors; however, their low transfection efficiency limits their clinical utility. To study the critical steps involved in an efficient transgene expression, there is a need for creative approaches that allow a systematic correlation between gene carrier structure and properties necessary for successful gene transfer. Using recombinant techniques a prototype vector comprised of tandem repeating units fused to a targeting moiety was biosynthesized to mediate gene transfer in mammalian cell lines. The carrier was designed to have the structure of (KHKHKHKHKK)6-FGF2 where lysine (K) residues would allow complexation with plasmid DNA, basic fibroblast growth factor (FGF2) to target cells over-expressing FGF2 receptors (FGFR), and histidine (H) residues to facilitate escape from the endosomal compartments. METHODS: The gene carrier was biosynthesized in E. coli, purified using a Ni-NTA column, characterized, complexed with pDNA, and the complexes were used to transfect NIH 3T3, T-47D and COS-1 mammalian cell types known to express FGFR. RESULTS: Results demonstrate the successful cloning and expression of the gene carrier with over 95% purity. The molecular weight of the gene carrier was determined by MALDI-TOF to be 27 402. Amino acid content analysis and Western blot confirmed the expression of the gene carrier in E. coli. The vector was able to condense pDNA, induce cell proliferation in NIH 3T3 fibroblasts, and mediate transgene expression in NIH 3T3, T-47D and COS-1 mammalian cell types. CONCLUSION: Genetic engineering techniques show promise for systematic investigation of structure-activity relationships of non-viral gene delivery vectors.

Recombinant polymers for cancer gene therapy: a minireview.

A major challenge for successful cancer gene therapy is the development of safe and effective gene delivery vectors. Gene delivery vectors can be viral or nonviral. Among nonviral vectors various polymeric vectors have shown potential in gene delivery. However, much work needs to be done in order to correlate polymer structure with gene release at the target site and transfection efficiency. This article is a brief introduction into cancer gene therapy, barriers and methods for gene transfer with emphasis on the applications of recombinant polymers for cancer gene therapy.

Transport mechanism(s) of poly (amidoamine) dendrimers across Caco-2 cell monolayers.

The objective of this research was to investigate the mechanism(s) of transport of generation 2 (G2) poly (amidoamine) dendrimers across Caco-2 cell monolayers. The contribution of an energy-dependent process such as adsorptive endocytosis was investigated by determining G2 permeability at 4 and 37 degrees C. The contribution of P-gp efflux to transport was examined by determining the apical to basolateral (AB) and basolateral to apical (BA) permeability of 14C-paclitaxel in presence of G2, and by determining AB and BA permeability of G2 in presence of paclitaxel. The permeability of G2 and 14C-mannitol was investigated in the presence of palmitoyl carnitine to determine the contribution of the paracellular pathway. Permeability of G2 at 4 degrees C was significantly (P<0.05) lower than that observed at 37 degrees C. AB and BA permeability of 14C-paclitaxel did not change in the presence of G2. AB and BA permeability of G2 did not change in the presence of paclitaxel. The permeability of G2 and 14C-mannitol increased significantly (P<0.05) in the presence of palmitoyl carnitine, and in addition, 14C-mannitol permeability was increased in presence of G2. The permeability of G2 across Caco-2 cell monolayers appears to involve a combination of paracellular transport and an energy-dependent process, possibly adsorptive endocytosis. G2 dendrimers do not appear to be substrates for the P-gp efflux system.

N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers for targeted delivery of 8-aminoquinoline antileishmanial drugs.

A challenge to successful chemotherapy of visceral leishmaniasis is the dose-limiting toxicity of antileishmanial agents. One approach to increase the efficacy and reduce the toxicity of these agents is to direct the drug to the phagolysosomes of the reticuloendothelial system (RES) where the leishmanial parasites reside. In this work a series of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-antileishmanial drug conjugates containing lysosomally degradable side chains and with or without sugar targeting moieties were synthesized, characterized and investigated for their in vivo efficacy in mice infected with Leishmania. An 8-aminoquinoline analog, namely 8-[(4-amino-1-methylbutyl)amino]-5-[3,4-dichlorophenoxy]-6-methoxy-4-methylquinoline (NPC1161) was used as a model antileishmanial agent. At 5 mg/kg body weight drug equivalent dose, all HPMA copolymer-drug conjugates which contained lysosomally degradable side chains showed significant in vivo antileishmanial activity (>99% inhibition), comparable to the activity of the free drug. At 2 mg dose, the same conjugates were significantly more effective (84-90% inhibition) than the free drug (67% inhibition). These results indicate the potential of lysosomotropic HPMA copolymers for the targeted delivery of antileishmanial compounds in the treatment of visceral leishmaniasis.

Extravasation of poly(amidoamine) (PAMAM) dendrimers across microvascular network endothelium.

PURPOSE: To study the influence of a controlled incremental increase in size and molecular weight of a series of poly(amidoamine) (PAMAM) dendrimers on their extravasation across the microvascular network endothelium. METHODS: A series of PAMAM dendrimers (generations 0-4) were fluorescently labeled using fluorescein isothiocyanate (FITC). Purification and fractionation of the fluorescently labeled polymers were done using size exclusion chromatography. The hamster cremaster muscle preparation was used as an in vivo model to study the extravasation process of the fluorescently labeled polymers. The extravasation process was visualized and recorded using intravital microscopy techniques. Analysis of the recorded experiments was done using Metamorph Imaging System. Extravasation of the fluorescently labeled polymers is reported in terms of their extravasation time (tau), i.e., the time needed for the fluorescence intensity in the interstitial tissue to reach 90% of the fluorescence intensity in the neighboring microvessels. RESULTS: Extravasation time (tau) describes the rate of microvascular extravasation of polymeric drug carriers across the microvascular endothelium into the interstitial tissue. Extravasation time (tau) of the studied PAMAM dendrimers showed size and molecular weight dependence. An increase in size and/or molecular weight of PAMAM dendrimers resulted in a corresponding exponential increase in the extravasation time (tau). CONCLUSIONS: Extravasation of PAMAM dendrimers across the microvascular endothelium showed size and molecular weight dependence. Results suggest that in addition to size and molecular weight, other physicochemical properties of polymeric drug carriers such as molecular geometry and charge may influence their microvascular extravasation. Systematic studies of the influence of the physico-chemical properties of polymeric drug carriers on their microvascular extravasation will aid in the design of novel macromolecular drug carriers with controlled extravasation profiles.

Molecular modeling of arginine-glycine-aspartic acid (RGD) analogs: relevance to transepithelial transport.

PURPOSE: The aim of this research is to model the effect of methylation on hydrogen bonding ability, surface area, polar surface area, volume, lipophilicity, charge, and cross-sectional diameters of a series of mono-, di-, and tri- methyl substituted analogs of arginine-glycine-aspartic acid (RGD) and compare these parameters to in vitro transport properties across Caco-2 monolayers. METHODS: Molecular modeling was used to investigate the structural parameters that may influence the transport properties of RGD and its methyl analogs at pH 7.4. Log P was experimentally determined using a potentiometric method and compared to cLogP. Transport studies were carried out using Caco-2 cell monolayers. RESULTS: Parameters such as polar and total surface area, volume, and Log P were found to vary with both the number and the sites of methyl substitution on the RGD molecule. The calculated as well as the experimental Log P values were found to be less than minus 2. The calculated maximum cross-sectional diameters ranged from 9 to 12 A. No detectable transport was noted. CONCLUSIONS: Results of our study indicate that in the design considerations for the development of new peptidomimetic RGD analogs with enhanced oral bioavailability, an important parameter to consider is the three dimensional conformation of the peptides which influences their hydrogen bonding ability, polarity and molecular geometry.

Transport of poly amidoamine dendrimers across Madin-Darby canine kidney cells.

The objective of this study was to determine the permeability of a series of poly amidoamine (PAMAM) dendrimers of generations 0-4 (G0-G4) across MDCK (Madin-Darby Canine Kidney) cell line. PAMAM dendrimers with incremental increase in size and molecular weight were labeled by fluorescein isothiocyanate (FITC) and the least polydisperse fractions were collected by size exclusion chromatography. MDCK cells were grown on Transwell filters for four days. The conjugates were detected by HPLC equipped with fluorescence detector. The permeability of the dendrimers across MDCK cells was determined in the apical to basolateral direction. The rank-order permeability of the PAMAM dendrimers was G4 >> G1 approximately G0 > G3 > G2. The permeability of mannitol in the presence of G4 increased by nine-fold. Results suggest that the transepithelial transport of PAMAM dendrimers is effected by both the polymer size, and the modulation of the cell membrane by the cationic dendrimers.

Novel site-specific chemical delivery system as a potential mydriatic agent: formation of phenylephrine in the iris-ciliary body from phenylephrone chemical delivery systems.

The objective of this study was to test the three novel ester derivatives of phenylephrone (isovaleryl, phenylacetyl, and pivalyl esters) as potential site-specific chemical delivery systems. The mydriatic effect and ocular distribution/metabolism of these compounds were studied by topical application to the eyes of normal rabbits. It was assumed that a reduction-hydrolysis sequence could produce the active phenylephrine in the iris-ciliary body tissues. All the derivatives showed a more pronounced mydriatic effect than that of phenylephrine, whereas phenylephrone was completely devoid of any mydriatic activity. Phenylacetyl ester was the most potent drug, with short duration of action, and showed maximum activity in the presence of 0.01% benzalkonium chloride without causing any visible irritation to the rabbit eye. Administration of the novel compounds to the eyes of the rabbits showed no traces of phenylephrine in the systemic circulation, contrary to topical administration of phenylephrine. Phenylephrone was detected in different compartments of the eye, whereas phenylephrine was present only in the iris-ciliary body tissues following administration of phenylacetyl ester. The conversion of phenylephrone esters to the active drug, phenylephrine, and thus their subsequent activity was dependent on the physicochemical characteristics of the drugs. The results suggest the potential use of phenylacetyl ester as a potent short-term mydriatic agent without systemic side effects.