David Stepensky

Novel Gastroretentive Dosage Forms: Evaluation of Gastroretentivity and Its Effect on Riboflavin Absorption in Dogs

AbstractPurpose. The purpose of this study was to design novel gastroretentive dosage forms (GRDFs) based on unfolding multilayer polymeric films, to investigate the mechanism of their gastroretentivity in dogs, and to assess the effect of compounding a narrow absorption window drug in a GRDF on the drugs absorption properties. Methods. Dosage forms (DFs) with different dimensions and mechanical properties were administered to beagle dogs with acidic buffer (pH=1.5), whose gastric retention time (GRT) was then determined by X-ray pictures. Concurrent administration of radiopaque markers was used to assess the effect of the GRDF and/or acidic buffer on GRT. The absorption of riboflavin from a prototype GRDF was compared with a nongastroretentive controlled-release DF and to an oral solution of the drug. Results. Large DFs (≥2.5 × 2.5 cm) containing rigid frame had prolonged GRT (>4 h). Administration of 400 mL of acidic buffer (or water) prolonged GRT whereas the GRDF did not cause additional prolongation. The extended absorption phase (>48 h) of riboflavin administered in a GRDF led to 4-fold increased bioavailability. Conclusion. The combination of large dimensions with rigidity produce gastroretentivity that can be used to improve absorption properties of a model of narrow absorption window drugs in the gastrointestinal tract.

Furosemide Pharmacokinetics and Pharmacodynamics following Gastroretentive Dosage Form Administration to Healthy Volunteers

The objective of this study was to evaluate the pharmacokinetic and pharmacodynamic properties of furosemide following gastroretentive dosage form (GRDF) administration. A furosemide (60 mg) GRDF, releasing the drug during 6 hours in vitro, or an immediate‐release tablet was administered to healthy male volunteers (N = 14) in a crossover design. Food and liquid intake were standardized; urine was collected, weighed, and assayed for furosemide and sodium concentrations. Pharmacokinetics of furosemide following the GRDF administration, as compared to the tablet, showed lower Cmax and indicated a prolonged absorption phase leading to longer mean residence time in the stomach. The sustained input of the drug significantly improved diuretic and natriuretic efficiencies during the first 5 hours and thereby increased the total effects measured over 24 hours. The unfolding controlled‐release GRDF of furosemide improved the pharmacodynamic actions due to the sustained absorption in the stomach and jejunum, which delayed the bodys counteractivity to the drug effect.

Bone as an effect compartment: Models for uptake and release of drugs

Abstract‘Bone-seeking agents’ are drugs characterised by high affinity for bone, and are disposed in bone for prolonged periods of time while maintaining remarkably low systemic concentrations. As a consequence, the bone becomes a reservoir for bone-seeking agents, and a site of both desirable and adverse effects, depending on the pharmacological activities of the specific agent. For some agents, significant systemic effects may also be produced following their prolonged release from bone, a process that is governed mostly by the rate of bone remodelling.This review covers the pharmacokinetic and pharmacodynamic features of bone-seeking agents with different pharmacological properties, including drugs (bisphosphonates, drug-bisphosphonate conjugates, radiopharmaceuticals and fluoride), bone markers (tetracycline, bone imaging agents) and toxins (lead, chromium, aluminium). In addition, drugs that do not possess bone-seeking properties but are used for therapy of bone diseases (such as antibacterials for treatment of osteomyelitis) are discussed, along with targeting of these drugs to the bone by conjugation to bone-seeking agents, local delivery systems, and other approaches.The pharmacokinetic and pharmacodynamic behaviour of bone-seeking agents is extremely complex due to heterogeneity in bone morphology and physiology. This complexity, accompanied by difficulties in human bone research caused by ethical and other limitations, gave rise to modelling approaches to study bone drug disposition. This review describes the pharmacokinetic models that have been proposed to describe the pharmacokinetic behaviour of bone-seeking agents and predict bone concentrations of these agents for different doses and patient populations. Models of different types (compartmental and physiologically based) and of different complexity have been applied, but their relevance to drug effects in the bone tissue is limited since they describe the behaviour of the ‘average’ drug molecule. Understanding of the cellular and molecular processes responsible for the heterogeneity of bone tissue will provide better comprehension of the influence of microenvironment on drug bone disposition and the resulting pharmacological response.

Solubility and Bioavailability of Stabilized Amorphous Calcium Carbonate

Since its role in the prevention of osteoporosis in humans was proven some 30 years ago, calcium bioavailability has been the subject of numerous scientific studies. Recent technology allowing the production of a stable amorphous calcium carbonate (ACC) now enables a bioavailability analysis of this unique form of calcium. This study thus compares the solubility and fractional absorption of ACC, ACC with chitosan (ACC‐C), and crystalline calcium carbonate (CCC). Solubility was evaluated by dissolving these preparations in dilute phosphoric acid. The results demonstrated that both ACC and ACC‐C are more soluble than CCC. Fractional absorption was evaluated by intrinsically labeling calcium carbonate preparations with 45Ca, orally administrated to rats using gelatin capsules. Fractional absorption was determined by evaluating the percentage of the administrated radioactive dose per milliliter that was measured in the serum, calcium absorption in the femur, and whole‐body retention over a 34‐hour period. Calcium serum analysis revealed that calcium absorption from ACC and ACC‐C preparations was up to 40% higher than from CCC, whereas retention of ACC and ACC‐C was up to 26.5% higher than CCC. Absorbed calcium in the femurs of ACC‐administrated rats was 30% higher than in CCC‐treated animals, whereas 15% more calcium was absorbed following ACC‐C treatment than following CCC treatment. This study demonstrates the enhanced solubility and bioavailability of ACC over CCC. The use of stable ACC as a highly bioavailable dietary source for calcium is proposed based on the findings of this study.

Delivery of proteins to the brain by bolaamphiphilic nano-sized vesicles.

Bolaamphiphilic cationic vesicles with acetylcholine (ACh) surface groups were investigated for their ability to deliver a model protein-bovine serum albumin conjugated to fluorescein isothiocyanate (BSA-FITC) across biological barriers in vitro and in vivo. BSA-FITC-loaded vesicles were internalized into cells in culture, including brain endothelial b.End3 cells, at 37 °C, but not at 4 °C, indicating an active uptake process. To examine if BSA-FITC-loaded vesicles were stable enough for in vivo delivery, we tested vesicle stability in whole serum. The half-life of cationic BSA-FITC-loaded vesicles with ACh surface groups that are hydrolyzed by choline esterase (ChE) was about 2 h, whereas the half-life of vesicles with similar surface groups, but which are not hydrolyzed by choline esterase (ChE), was over 5 h. Pyridostigmine, a choline esterase inhibitor that does not penetrate the blood-brain barrier (BBB), increased the stability of the ChE-sensitive vesicles to 6 h but did not affect the stability of vesicles with ACh surface groups that are not hydrolyzed by ChE. Following intravenous administration to pyridostigmine-pretreated mice, BSA-FITC encapsulated in ChE-sensitive vesicles was distributed into various tissues with marked accumulation in the brain, whereas non-encapsulated (free) BSA-FITC was detected only in peripheral tissues, but not in the brain. These results show that cationic bolaamphiphilic vesicles with ACh head groups are capable of delivering proteins across biological barriers, such as the cell membrane and the blood-brain barrier (BBB). Brain ChE activity destabilizes the vesicles and releases the encapsulated protein, enabling its accumulation in the brain.

O-glycosylated versus non-glycosylated MUC1-derived peptides as potential targets for cytotoxic immunotherapy of carcinoma

Due to the fact that many cellular proteins are extensively glycosylated, processing and presentation mechanisms are expected to produce a pool of major histocompatibility complex (MHC) class I‐bound protein‐derived peptides, part of which retain sugar moieties. The immunogenic properties of the presented glycosylated peptides in comparison to their non‐glycosylated counterparts have not been determined clearly. We assessed the cellular immunogenicity of MUC1 (mucin)‐derived peptides O‐glycosylated with a Tn epitope (GalNAc) using HLA‐A*0201 single chain (HHD)‐transfected cell lines and transgenic mice. For part of the compounds Tn moiety did not interfere with the HLA‐A*0201 binding. Moreover, part of the glycopeptides elicited effective cytotoxic responses, indicating recognition of the glycopeptide‐HLA‐A*0201 complex by the T cell receptor (TCR) and subsequent cytotoxic T lymphocyte (CTL) activation. The CTLs exhibited a substantial degree of cross‐reactivity against target cells loaded with glycosylated and non‐glycosylated forms of the same peptide. The studied (glyco)peptides showed cellular immunogenicity in both MUC1‐HHD and HHD mice and induced effective lysis of (glyco)peptide‐loaded target cells in CTL assays. However, the elicited CTLs did not induce selective lysis of human MUC1‐expressing murine cell lines. Moreover, immunization with (glyco)peptide‐loaded dendritic cells (DCs) did not induce significant immunotherapeutic effects. We conclude that Tn glycosylated MUC1‐derived peptides can be presented by MHC class I molecules, and may be recognized by specific TCR molecules resulting in cytotoxic immune responses. However, the studied glycopeptides did not offer significant benefit as targets for cytotoxic immune response due apparently to (a) cross‐reactivity of the elicited CTLs against the glycosylated and non‐glycosylated forms of the same peptide and (b) low abundance of glycopeptides on tumour target cells.

Mode of administration-dependent pharmacokinetics of bisphosphonates and bioavailability determination

We investigated the influence of mode of administration on the pharmacokinetics of a clinically used bisphosphonate, pamidronate, and of suberoylbisphosphonate (SuBP), a novel bisacylphosphonate of the P-CO-(C)(n)-CO-P type, in rats. Serum drug levels and tissue disposition were determined following administration of the drugs by different modes: intravenous bolus (iso-osmotic and hypo-osmotic solutions), continuous intravenous infusion, and peroral administration. Results of the study indicate that the disposition of the bisphosphonates in soft tissue (liver, kidney and spleen) was dependent on route and rate of drug administration, and on the osmoticity of the vehicle. Consequently, main pharmacokinetic parameters (AUC, CL, and V(ss)) were influenced by the mode of drug administration, precluding accurate determination of bioavailability from AUC values. On the other hand, bone and urine bisphosphonate accumulation were considerably less dependent on mode of administration, and, therefore, are recommended for bioavailability calculation.

Imaging cancer cells expressing the folate receptor with carbon dots produced from folic acid

Development of new imaging tools for cancer cells in vitro and in vitro is important for advancing cancer research, elucidating drug effects upon cancer cells, and studying cellular processes. We showed that fluorescent carbon dots (C‐dots) synthesized from folic acid can serve as an effective vehicle for imaging cancer cells expressing the folate receptor on their surface. The C‐dots, synthesized through a simple one‐step process from folic acid as the carbon source, exhibited selectivity towards cancer cells displaying the folate receptor, making such cells easily distinguishable in fluorescence microscopy imaging. Biophysical measurements and competition experiments both confirmed the specific targeting and enhanced uptake of C‐dots by the folate receptor‐expressing cells. The folic acid‐derived C‐dots were not cytotoxic, and their use in bioimaging applications could aid biological studies of cancer cells, identification of agonists/antagonists, and cancer diagnostics.

Synthesis and preclinical pharmacology of 2-(2-aminopyrimidinio) ethylidene-1,1-bisphosphonic acid betaine (ISA-13-1)-a novel bisphosphonate.

AbstractPurpose. To validate our hypothesis that a bisphosphonate (BP) having a nitrogen-containing heterocyclic ring on the side chain, and with no hydroxyl on the geminal carbon would possess increased activity, and better oral bioavailability due to enhanced solubility of its calcium complexes/salts and weaker Ca chelating properties. Methods. A novel BP, 2-(2-aminopyrimidinio)ethylidene-l,l-bisphosphonic acid betaine (ISA-13-1) was synthesized. The physicochemical properties and permeability were studied in vitro. The effects on macrophages, bone resorption (young growing rat model), and tumor-induced osteolysis (Walker carcinosarcoma) were studied in comparison to clinically used BPs. Results. The solubility of the Ca salt of ISA-13-1 was higher, and the log βCa: BP stability constant and the affinity to hydroxyapatite were lower than those of alendronate and pamidronate. ISA-13-1 exhibited effects similar to those of alendronate on bone volume, on bone osteolysis, and on macrophages, following delivery by liposomes. ISA-13-1 was shown to have 1.5−1.7 times better oral absorption than the other BPs with no deleterious effects on the tight junctions of intestinal tissue. Conclusions. The similar potency to clinically used BPs, the increased oral absorption as well as the lack of effect on tissue tight junction of ISA-13-1 warrant its further consideration as a potential drug for bone diseases.

Delivery of analgesic peptides to the brain by nano-sized bolaamphiphilic vesicles made of monolayer membranes

Inefficient drug delivery to the brain is a major obstacle for pharmacological management of brain diseases. We investigated the ability of bolavesicles - monolayer membrane vesicles self-assembled from synthetic bolaamphiphiles that contain two hydrophilic head groups at each end of a hydrophobic alkyl chain - to permeate the blood-brain barrier and to deliver the encapsulated materials into the brain. Cationic vesicles with encapsulated kyotorphin and leu-enkephalin (analgesic peptides) were prepared from the bolalipids GLH-19 and GLH-20 and studied for their analgesic effects in vivo in experimental mice. The objectives were to determine: (a) whether bolavesicles can efficiently encapsulate analgesic peptides, (b) whether bolavesicles can deliver these peptides to the brain in quantities sufficient for substantial analgesic effect, and to identify the bolavesicle formulation/s that provides the highest analgetic efficiency. The results indicate that the investigated bolavesicles can deliver analgesic peptides across the blood-brain barrier and release them in the brain in quantities sufficient to elicit efficient and prolonged analgesic activity. The analgesic effect is enhanced by using bolavesicles made from a mixture the bolas GLH-19 (that contains non-hydrolyzable acetylcholine head group) and GLH-20 (that contains hydrolysable acetylcholine head group) and by incorporating chitosan pendants into the formulation. The release of the encapsulated materials (the analgesic peptides kyotorphin and leu-enkephalin) appears to be dependent on the choline esterase (ChE) activity in the brain vs. other organs and tissues. Pretreatment of experimental animals with pyridostigmine (the BBB-impermeable ChE inhibitor) enhances the analgesic effects of the studied formulations. The developed formulations and the approach for their controlled decapsulation can serve as a useful modality for brain delivery of therapeutically-active compounds.