A renaissance in marine pharmacology: From preclinical curiosity to clinical reality

Marine pharmacology, the pharmacology of marine natural products, has been for some time more associated with marine natural products chemistry rather than mainstay pharmacology. However, in recent years a renaissance has occurred in this area of research, and has seen the US Food & Drug Administration (FDA) approval in 2004 of Prialt^® (ziconotide, ω-conotoxin MVIIA) the synthetic equivalent of a conopeptide found in marine snails, used for the management of severe chronic pain. Furthermore Yondelis^® (trabectedin, ET-743) an antitumor agent discovered in a marine colonial tunicate, and now produced synthetically, receiving Orphan Drug designation from the European Commission (EC) and FDA for soft tissue sarcomas and ovarian cancer and its registration in 2007 in the EU for the treatment of soft tissue sarcoma. The approval/marketing of so few marine natural products has come after many years of research primarily by the academic community and the sporadic involvement of major pharmaceutical companies. This commentary, through the opinions provided by several leaders in the marine natural products field, will examine the potential reasons and perceptions from both the academic and pharmaceutical communities regarding the development of marine natural products as viable therapeutic entities.     

In situ formation of nanoparticles upon dispersion of melt extrudate formulations in aqueous medium assessed by asymmetrical flow field-flow fractionation

In recent years melt extrudates (e.g. Meltrex^®) have proven to be a promising formulation tool for poorly water-soluble and poorly bioavailable drugs. During the hot-melt extrusion process solid dispersions are formed. For several of these formulations improved bioavailabilities have been reported; the mechanism behind, however is still not very well understood. The aim of this study was to investigate whether solid dispersions prepared by melt extrusion upon dispersion in aqueous medium form particles and/or supramolecular assemblies. The formulation investigated here contained the human immunodeficiency virus (HIV) protease inhibitors lopinavir and ritonavir, polyvinylpyrrolidone–vinyl acetate copolymer (Kollidon^® VA64), sorbitan monolaurate (Span^® 20) and hydrophilic fumed silica (Aerosil^® 200). The aqueous dispersions originating from both, API-containing and placebo formulation were investigated using photon correlation spectroscopy (PCS) and asymmetrical flow field-flow fractionation (AsFlFFF) with subsequent online multi-angle light-scattering (MALS) particle size analysis. The content of both APIs in the AsFlFFF-fractions was quantified using high performance liquid chromatography–mass spectrometry.     

A comparative pH-dissolution profile study of selected commercial levothyroxine products using inductively coupled plasma mass spectrometry

Levothyroxine (T4) is a narrow therapeutic index drug with classic bioequivalence problem between various available products. Dissolution of a drug is a crucial step in its oral absorption and bioavailability. The dissolution of T4 from three commercial solid oral dosage forms: Synthroid^® (SYN), generic levothyroxine sodium by Sandoz Inc. (GEN) and Tirosint^® (TIR) was studied using a sensitive ICP-MS assay. All the three products showed variable and pH-dependent dissolution behaviors. The absence of surfactant from the dissolution media decreased the percent T4 dissolved for all the three products by 26-95% (at 30 min). SYN dissolution showed the most pH dependency, whereas GEN and TIR showed the fastest and highest dissolution, respectively. TIR was the most consistent one, and was minimally affected by pH and/or by the presence of surfactant. Furthermore, dissolution of T4 decreased considerably with increase in the pH, which suggests a possible physical interaction in patients concurrently on T4 and gastric pH altering drugs, such as proton pump inhibitors. Variable dissolution of T4 products can, therefore, impact the oral absorption and bioavailability of T4 and may result in bioequivalence problems between various available products.     

The multikinase inhibitor axitinib is a potent inhibitor of human CYP1A2

The tyrosine kinase inhibitors (TKIs) and multikinase inhibitors (MKIs) are oncology drugs of increasing importance that have improved the treatment of multiple tumors types. In some patients these agents produce adverse effects, including pharmacokinetic drug–drug interactions, due to cytochrome P450 (CYP) inhibition. Information on the propensity of the drugs to elicit such effects often only becomes evident as the drugs enter clinical use. The present study assessed 18 kinase inhibitors (1 and 50 μM) for the inhibition of major drug metabolizing CYPs 1A2, 2C9, 2D6 and 3A4 in human liver microsomes. Most TKIs and MKIs inhibited CYP reactions at the higher concentration but axitinib also potently inhibited CYP1A2-dependent 7-ethoxyresorufin O-deethylation activity at the lower concentration. Kinetic analyses of CYP1A2 inhibition by axitinib were undertaken in microsomes and found a K_i of 0.11 ± 0.01 μM, which was 7.5-fold lower than the K_m for 7-ethoxyresorufin oxidation (0.83 ± 0.06 μM); the inhibition mechanism was linear-mixed. From computational modeling two potential binding modes for axitinib were identified in the active site of CYP1A2: one in which the oxidizable axitinib thioether sulfur atom is within ∼4.45 Å of the CYP1A2 heme, and is likely to favor biotransformation of the drug, and a second in which the pyridine moiety is in proximity to the heme, which may contribute to inhibition. The applicability of these findings to potential pharmacokinetic interactions in patients during axitinib treatment should now be assessed.     

Enhancement of griseofulvin release from liquisolid compacts

The potential of hydrophilic aerogel formulations and liquisolid systems to improve the release of poorly soluble drugs was investigated using griseofulvin as model drug. The in vitro release rates of this drug formulated as directly compressed tablets containing crystalline griseofulvin were compared to aerogel tablets with the drug adsorbed onto hydrophilic silica aerogel and to liquisolid compacts containing the drug dissolved or suspended in PEG 300. Furthermore, the commonly used carrier and coating materials in liquisolid systems Avicel® and Aerosil® were replaced by Neusilin®, an amorphous magnesium aluminometasilicate with an extremely high specific surface area of 339 m²/g to improve the liquisolid approach.Both the liquisolid compacts containing the drug dissolved in PEG 300 and the aerogel tablets showed a considerably faster drug release than the directly compressed tablets. With liquisolid compacts containing the drug suspended in PEG 300, the release rate increased with rising fraction of dissolved drug in the liquid portion. It could be shown that Neusilin® with its sevenfold higher liquid adsorption capacity than the commonly used Avicel® and Aerosil® allows the production of liquisolid formulations with lower tablet weights.     

Lapatinib and erlotinib are potent reversal agents for MRP7 (ABCC10)-mediated multidrug resistance

In recent years, a number of TKIs (tyrosine kinase inhibitors) targeting epidermal growth factor receptor (EGFR) family have been synthesized and some have been approved for clinical treatment of cancer by the FDA. We recently reported a new pharmacological action of the 4-anilinoquinazoline derived EGFR TKIs, such as lapatinib (Tykerb^®) and erlotinib (Tarceva^®), which significantly affect the drug resistance patterns in cells expressing the multidrug resistance (MDR) phenotype. Previously, we showed that lapatinib and erlotinib could inhibit the drug efflux function of P-glycoprotein (P-gp, ABCB1) and ABCG2 transporters. In this study, we determined if these TKIs have the potential to reverse MDR due to the presence of the multidrug resistance protein 7 (MRP7, ABCC10). Our results showed that lapatinib and erlotinib dose-dependently enhanced the sensitivity of MRP7-transfected HEK293 cells to several established MRP7 substrates, specifically docetaxel, paclitaxel, vinblastine and vinorelbine, whereas there was no or a less effect on the control vector transfected HEK293 cells. [³H]-paclitaxel accumulation and efflux studies demonstrated that lapatinib and erlotinib increased the intracellular accumulation of [³H]-paclitaxel and inhibited the efflux of [³H]-paclitaxel from MRP7-transfected cells but not in the control cell line. Lapatinib is a more potent inhibitor of MRP7 than erlotinib. In addition, the Western blot analysis revealed that both lapatinib and erlotinib did not significantly affect MRP7 expression. We conclude that the EGFR TKIs, lapatinib and erlotinib reverse MRP7-mediated MDR through inhibition of the drug efflux function, suggesting that an EGFR TKI based combinational therapy may be applicable for chemotherapeutic practice clinically.     

Albendazole Generics—A Comparative In Vitro Study

Purpose. We sought to determine whether disintegration and dissolution behavior differs among various albendazole generic formulations obtained from third world countries and to compare them with the innovator's product. Methods. Dissolution behavior of various albendazole formulations was studied with USP Apparatus 2 in SGF_sp and in a modified SGF_sp which contained 0.1% of the nonionic surfactant Triton^® × 100. Disintegration was tested according to the European Pharmacopoeia. Results. Dissolution experiments in SGF_sp showed a wide range in rate and extent of albendazole dissolution. The innovator product released 81 percent within two hours, a profile matched by only one other formulation. For other formulations 32 to 64% was released within two hours. Use of a modified SGF_sp, containing 0.1% Triton^® × 100 to simulate the surface tension of gastric juice, resulted in less discrimination between products. The innovator product again showed the fastest and most complete dissolution, with ninety percent released within two hours. The generic formulations released between 67 and 82%, except for one formulation which achieved only 43% release. The results in SGF_sp plus Triton^® × 100 may be more meaningful than in SGF_sp, since the surface tension of the medium is closer to the physiological value. All formulations passed the disintegration test according to the European Pharmacopoeia, with disintegration times ranging from 2.5 to 11 minutes. Conclusions. Generic albendazole products vary widely in their dissolution behavior. Differences among products were greater in SGF_sp than in SGF_sp plus Triton^® × 100. These differences were not reflected in the disintegration behavior of the products.     

Two natural products, trans-phytol and (22E)-ergosta-6,9,22-triene-3β,5α,8α-triol,inhibit the biosynthesis of estrogen in human ovarian granulosa cells by aromatase (CYP19)

Aromatase is the only enzyme in vertebrates to catalyze the biosynthesis of estrogens. Although inhibitors of aromatase have been developed for the treatment of estrogen-dependent breast cancer, the whole-body inhibition of aromatase causes severe adverse effects. Thus, tissue-selective aromatase inhibitors are important for the treatment of estrogen-dependent cancers. In this study, 63 natural products with diverse structures were examined for their effects on estrogen biosynthesis in human ovarian granulosa-like KGN cells. Two compounds—trans-phytol (SA-20) and (22E)-ergosta-6,9,22-triene-3β,5α,8α-triol (SA-48)—were found to potently inhibit estrogen biosynthesis (IC₅₀: 1 μM and 0.5 μM, respectively). Both compounds decreased aromatase mRNA and protein expression levels in KGN cells, but had no effect on the aromatase catalytic activity in aromatase-overexpressing HEK293A cells and recombinant expressed aromatase. The two compounds decreased the expression of aromatase promoter I.3/II. Neither compound affected intracellular cyclic AMP (cAMP) levels, but they inhibited the phosphorylation or protein expression of cAMP response element-binding protein (CREB). The effects of these two compounds on extracellular regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinases (MAPKs), and AKT/phosphoinositide 3-kinase (PI3K) pathway were examined. Inhibition of p38 MAPK could be the mechanism underpinning the actions of these compounds. Our results suggests that natural products structurally similar to SA-20 and SA-48 may be a new source of tissue-selective aromatase modulators, and that p38 MAPK is important in the basal control of aromatase in ovarian granulosa cells. SA-20 and SA-48 warrant further investigation as new pharmaceutical tools for the prevention and treatment of estrogen-dependent cancers.     

Establishing the optimal nebulization system for paclitaxel,docetaxel, cisplatin,carboplatin and gemcitabine: Back to drawing the residual cup

Background

Chemotherapy drugs have still the major disadvantage of non-specific cytotoxic effects. Although, new drugs targeting the genome of the tumor are already in the market, doublet chemotherapy regimens still remain the cornerstone of lung cancer treatment. Novel modalities of administration are under investigation such as; aerosol, intratumoral and intravascular.

Materials and methods

In the present study five chemotherapy drugs; paclitaxel, docetaxel, gemcitabine, carboplatin and cisplatin were nebulized with three different jet nebulizers (Maxineb^®, Sunmist^®, Invacare^®) and six different residual cups at different concentrations. The purpose of the study was to identify the “ideal” combination of nebulizer-residual cup design-drug–drug loading for a future concept of aerosol chemotherapy in lung cancer patients. The Mastersizer^® 2000 was used to evaluate the aerosol droplet mass median aerodynamic diameter.

Results

The drug, nebulizer and residual cup design greatly influences the producing droplet size (p < 0.005, in each case). However; the design of the residual cup is the most important factor affecting the produced droplet size (F = 834.6, p < 0.001). The drug loading plays a vital role in the production of the desired droplet size (F = 10.42, p < 0.001). The smallest droplet size was produced at 8 ml loading (1.26 μm), while it remained the same at 2, 4 and 6 mls of drug loading.

Conclusion

The ideal nebulizer would be Maxineb^®, with a large residual cup (10 ml maximum loading capacity) and 8 mls loading and the drug with efficient pulmonary deposition would be docetaxel.     

Targeting the proteasome pathway

Background: The ubiquitin–proteasome pathway functions as a main pathway in intracellular protein degradation and plays a vital role in almost all cellular events. Various inhibitors of this pathway have been developed for research purposes. The recent approval of bortezomib (PS-341, Velcade^®), a proteasome inhibitor, for the treatment of multiple myeloma has opened the way to the discovery of drugs targeting the proteasome and other components of the ubiquitin–proteasome pathway. Objectives: We review the current understanding of the ubiquitin–proteasome pathway and inhibitors targeting this pathway, including proteasome inhibitors, as candidate drugs for chemical therapy. Methods: Preclinical and clinical data for inhibitors of the proteasome and the ubiquitin–proteasome pathway are discussed. Conclusions: The proteasome and other members in the ubiquitin–proteasome pathway have emerged as novel therapeutic targets.     

Dissolution profiles of mesalazine formulationsin vitro

In vitro dissolution profiles of three controlled-release mesalazine formulations were determined at pH 1.0, 6.0 and 7.5. A closed-column type dissolution apparatus was used. A reproducible gradual dissolution profile was seen for Pentasa^® at all pH values. Dissolution starts immediately and is complete after 20 h. Dissolution profiles at pH 1 and pH 7.5 are much alike and dissolution is faster than at pH 6. The behaviour of Asacol^® at different pH values corresponds with the expectations: no release at pH 6 and pH 1, fast release at pH 7.5. Dissolution starts after 1 h and is complete after 3 h. Mesalazine release from Salofalk^® tablets at pH 7.5 and pH 6.0 starts after 2 and 3 h, respectively, and is complete after 5 and 10 h. However, after a long lag-time (10 h) mesalazine is also released from Salofalksu^® tablets at pH 1 and dissolution is complete after 23 h.     

Chiral separations in normal-phase liquid chromatography: Enantioselectivity of recently commercialized polysaccharide-based selectors. Part II. Optimization of enantioselectivity

Earlier, a set of pharmaceuticals with different chemical structures has been used to evaluate the enantioselectivity of four recently commercialized polysaccharide-based chiral stationary phases, Lux^® Cellulose-1/Sepapak^® 1, Lux^® Cellulose-2/Sepapak^® 2, Lux^® Amylose-2/Sepapak^® 3 and Lux Cellulose-4/Sepapak^® 4 and of three Daicel columns, Chiralpak^® AD-H, Chiralcel^® OD-H and Chiralcel^® OJ-H, using the screening conditions of an existing generic separation strategy in normal-phase liquid chromatography (NPLC). In this study, the applicability of the optimization steps of the existing separation strategy was examined using 44 drugs (70 optimization cases) representing the three possible resolution situations that occur after screening. Optimizations are demonstrated by modifying parameters such as polar modifier percentages, temperatures, flow rates and additives concentration. Changing the percentage of polar modifier was found to have the largest influence on the resolution. The resolution, peak shape and the analysis time were nicely improved for 49/70 cases (70%) after the application of the original optimization steps. The introduction of some modifications to the original optimization increased this number from 49 to 62 cases, i.e. from 70% to 88.6%. Finally, an updated generic separation strategy in NPLC was proposed.     

Comparison of the in vitro dissolution properties and in vivo steady-state pharmacokinetics of two sustained-release theophylline preparations

The sustained-release properties and relative bioavailability of Theolin^® Retard and Pharphylline^® Retard were studied in eight healthy adults after treatment for five days with twice daily 450 mg, respectively 425 mg. During the day-time dosing interval on the fourth and fifth day theophylline plasma concentrations were assayed by HPLC. After intake of Theolin^® Retard, minimum theophylline plasma concentrations were significantly higher, fluctuations in theophylline plasma concentrations were significantly smaller andt ₇₅ (the period within a dosing interval during which the plasma concentration exceeds 75% of the maximal concentration) was significantly longer than after Pharphylline^® Retard. Maximal concentrations and AUC values were not significantly different. For both products the plasma concentration time-curves on day 5 were significantly lower than on day 4.In vitro dissolution tests confirmed the more sustained release of theophylline from Theolin^® Retard. These results indicate an equal extent of absorption from the two products but better sustained-release properties for Theolin^® Retard.     

A comparative study of the physicochemical properties of iron isomaltoside 1000 (Monofer®), a new intravenous iron preparation and its clinical implications

The treatment of iron deficiency anemia with polynuclear iron formulations is an established therapy in patients with chronic kidney disease but also in other disease areas like gastroenterology, cardiology, oncology, pre/post operatively and obstetrics’ and gynecology. Parenteral iron formulations represent colloidal systems in the lower nanometer size range which have traditionally been shown to consist of an iron core surrounded by a carbohydrate shell. In this publication, we for the first time describe the novel matrix structure of iron isomaltoside 1000 which differs from the traditional picture of an iron core surrounded by a carbohydrate. Despite some structural similarities between the different iron formulations, the products differ significantly in their physicochemical properties such as particle size, zeta potential, free and labile iron content, and release of iron in serum. This study compares the physiochemical properties of iron isomaltoside 1000 (Monofer®) with the currently available intravenous iron preparations and relates them to their biopharmaceutical properties and their approved clinical applications. The investigated products encompass low molecular weight iron dextran (CosmoFer®), sodium ferric gluconate (Ferrlecit®), iron sucrose (Venofer®), iron carboxymaltose (Ferinject®/Injectafer®), and ferumoxytol (Feraheme®) which are compared to iron isomaltoside 1000 (Monofer®). It is shown that significant and clinically relevant differences exist between sodium ferric gluconate and iron sucrose as labile iron formulations and iron dextran, iron carboxymaltose, ferumoxytol, and iron isomaltoside 1000 as stable polynuclear formulations. The differences exist in terms of their immunogenic potential, safety, and convenience of use, the latter being expressed by the opportunity for high single-dose administration and short infusion times. Monofer is a new parenteral iron product with a very low immunogenic potential and a very low content of labile and free iron. This enables Monofer, as the only IV iron formulation, to be administered as a rapid high dose infusion in doses exceeding 1000 mg without the application of a test dose. This offers considerable dose flexibility, including the possibility of providing full iron repletion in a single infusion (one-dose iron repletion).     

Kinase inhibitors as drugs for chronic inflammatory and immunological diseases: progress and challenges

At the time of writing, there are seven marketed kinase inhibitor drugs. The first kinase inhibitor, imatinib mesilate (Gleevec^®, Novartis), came to market in 2001, an inhibitor of the breakpoint cluster region (BCR)/Abelson murine leukemia oncogene homolog (ABL) fusion, platelet-derived growth factor (PDGF) receptor, and c-kit kinases. The most recent kinase inhibitor to come to market, disatinib (Sprycel^®, Bristol-Myers Squibb), acts on c-SRC, ABL and Bruton's tyrosine kinase. To date, kinase inhibitor drugs are approved for oncology and demonstrate that it is possible to develop compounds with relative selectivity for the target kinase against the broader kinome. However, the use of kinase inhibitors in chronic inflammatory and immunologic diseases may require greater selectivity for the target kinase. This review addresses the opportunities and challenges of kinase inhibition as a therapeutic approach in chronic immune and inflammatory disease.     

Practical formulation and process development of freeze-dried products

Freeze-drying science and technology continues to evolve and increase in importance because of the emergence of biotechnology drugs that are too unstable to be commercially available as ready-to-use solutions. As more new drug compounds need to be developed as freeze-dried products, this mini-review article provides practical guidance and commentary on the latest literature articles on formulation and process development of freeze-dried products. This article contains a table that provides the quantitative formulations of all commercial freeze-dried protein pharmaceutical products through 2004.     

Evaluation of the multipotent character of human foreskin-derived precursor cells

In the present study, the trilineage differentiation capacity of human foreskin-derived precursor cells (hSKP) was evaluated upon exposure to various (non)commercial (i and ii) ectodermal, (iii) mesodermal and (iv) endodermal differentiation media.

(i)

Upon sequential exposure of the cells to keratinocyte growth (CnT-07® or CnT-057®) and differentiation (CnT-02® or Epilife®) media, keratinocyte-like cells (filaggrin⁺/involucrin⁺) were obtained. The preferred keratinocyte differentiation strategy was exposure to CnT-07®.

(ii)

When hSKP were subsequently exposed to NeuroCult® media, cells underwent a weak neuro-ectodermal differentiation expressing nestin, myelin binding protein (MBP), vimentin and alpha-foetoprotein (AFP). Sequential exposure to NPMM® and NPDM® generated cells with an inferior neuro-ectodermal phenotype (nestin⁺/vimentin⁺/MBP⁻/AFP⁻).

(iii)

Upon exposure of hSKP to insulin-transferrin-selenite (ITS) and dexamethasone, small lipid droplets were observed, suggesting their differentiation potential towards adipocyte-like cells.

(iv)

Finally, after sequential exposure to hepatogenic growth factors and cytokines, an immature hepatic cell population was generated. The presence of pre-albumin suggests that a sequential exposure strategy is here superior to a cocktail approach.

In summary, a considerable impact of different (non)commercial media on the lineage-specific differentiation efficiency of hSKP is shown. In addition, we demonstrate here for the first time that, in a suitable keratinocyte stimulating micro-environment, hSKP can generate keratinocyte-like progeny in vitro.     

Curcumin-induced degradation of PKCδ is associated with enhanced dentate NCAM PSA expression and spatial learning in adult and aged Wistar rats

Polysialylation of the neural cell adhesion molecule (NCAM PSA) is necessary for the consolidation processes of hippocampus-based learning. Previously, we have found inhibition of protein kinase C delta (PKCδ) to be associated with increased polysialyltransferase (PST) activity, suggesting inhibitors of this kinase might ameliorate cognitive deficits. Using a rottlerin template, a drug previously considered an inhibitor of PKCδ, we searched the Compounds Available for Purchase (CAP) database with the Accelrys^® Catalyst programme for structurally similar molecules and, using the available crystal structure of the phorbol-binding domain of PKCδ, found that diferuloylmethane (curcumin) docked effectively into the phorbol site. Curcumin increased NCAM PSA expression in cultured neuro-2A neuroblastoma cells and this was inversely related to PKCδ protein expression. Curcumin did not directly inhibit PKCδ activity but formed a tight complex with the enzyme. With increasing doses of curcumin, the Tyr¹³¹ residue of PKCδ, which is known to direct its degradation, became progressively phosphorylated and this was associated with numerous Tyr¹³¹-phospho-PKCδ fragments. Chronic administration of curcumin in vivo also increased the frequency of polysialylated cells in the dentate infragranular zone and significantly improved the acquisition and consolidation of a water maze spatial learning paradigm in both adult and aged cohorts of Wistar rats. These results further confirm the role of PKCδ in regulating PST and NCAM PSA expression and provide evidence that drug modulation of this system enhances the process of memory consolidation.     

Exploiting p70 S6 kinase as a target for ovarian cancer

Introduction: The p70 S6 kinase (p70^S6K) is frequently active in ovarian and a wide range of cancer types, and it has a crucial role in several processes considered hallmarks of cancer. Therefore, blocking p70^S6K expression or activity may present a promising strategy for anticancer treatment.

Areas covered: The current understanding of the molecular mechanisms that govern p70^S6K regulation as well as its tumorigenic effects, which are involved in the initiation and progression in ovarian cancer, in particular the emerging new role of p70^S6K in cell migration, which is a prerequisite of tumor metastasis. The p70^S6K cellular substrates and/or interacting proteins. The current state of drugs that target this kinase, either alone or in combination with other targeted agents.

Expert opinion: Targeting p70^S6K through the use of small-molecule inhibitors, microRNAs and natural compounds may represent a beneficial new avenue for cancer therapy and opens new areas of investigation in p70^S6K biology.