Evaluation of dendrimer SPL7013, a lead microbicide candidate against herpes simplex viruses

Dendrimers are a novel class of polyanionic macromolecules with broad-spectrum antiviral activities and minimal toxicities. A new generation of amide dendrimer, SPL7013, was evaluated as a lead microbicide candidate against herpes simplex viruses (HSV). The plaque reduction assays showed that the 50% effective concentrations (EC(50)) determined by pre-treatment of cells were 2.0 microg/ml for HSV-1 and 0.5 microg/ml for HSV-2. Inhibitory effects were also observed on HSV-infected cells with EC(50)s of 6.1 microg/ml for HSV-1 and 3.8 microg/ml for HSV-2. These are the mean values from the test results of six batches of SPL7013. SPL7013 was also shown to be equally potent against HSV drug-resistant strains. SPL7013 completely inhibited viral adsorption to Vero cells at concentrations of higher than 3 microg/ml. Analyzed by a LightCycler assay after treatment of HSV-infected cells for 17 h, SPL7013 showed strong inhibition of HSV DNA synthesis with EC(50)s of approximately 6.2 and 2.0 microg/ml for HSV-1 and HSV-2, respectively. SPL7013 retained its anti-HSV activity even after treatment at acidic pHs 3.0 and 4.0 for 2 h. The presence of 10% human serum proteins did not affect the anti-HSV activity of SPL7013. SPL7013 was not toxic to Vero cells up to the highest concentration tested (10,000 microg/ml). Effects on cell proliferation were tested on two epithelial cell lines in both stationary and dividing phases. The 50% cytotoxic concentrations (CC(50)) in all cases were greater than 10,000 microg/ml. Our data indicate that SPL7013 is a promising candidate for development as a vaginal microbicide and a therapeutic agent.     

Virucidal activity of the dendrimer microbicide SPL7013 against HIV-1

Topical microbicides for use by women to prevent the transmission of human immunodeficiency virus (HIV) and other sexually transmitted infections are urgently required. Dendrimers are highly branched nanoparticles being developed as microbicides. SPL7013 is a dendrimer with broad-spectrum activity against HIV type I (HIV-1) and -2 (HIV-2), herpes simplex viruses type-1 (HSV-1) and -2 (HSV-2) and human papillomavirus. SPL7013 [3% (w/w)] has been formulated in a mucoadhesive carbopol gel (VivaGel?) for use as a topical microbicide. Previous studies showed that SPL7013 has similar potency against CXCR4-(X4) and CCR5-using (R5) strains of HIV-1 and that it blocks viral entry. However, the ability of SPL7013 to directly inactivate HIV-1 is unknown. We examined whether SPL7013 demonstrates virucidal activity against X4 (NL4.3, MBC200, CMU02 clade EA and 92UG046 clade D), R5 (Ba-L, NB25 and 92RW016 clade A) and dual-tropic (R5X4; MACS1-spln) HIV-1 using a modified HLA-DR viral capture method and by polyethylene glycol precipitation. Evaluation of virion integrity was determined by ultracentrifugation through a sucrose cushion and detection of viral proteins by Western blot analysis. SPL7013 demonstrated potent virucidal activity against X4 and R5X4 strains, although virucidal activity was less potent for the 92UG046 X4 clade D isolate. Where potent virucidal activity was observed, the 50% virucidal concentrations were similar to the 50% effective concentrations previously reported in drug susceptibility assays, indicating that the main mode of action of SPL7013 is by direct viral inactivation for these strains. In contrast, SPL7013 lacked potent virucidal activity against R5 HIV-1 strains. Evaluation of the virucidal mechanism showed that SPL7013-treated NL4.3, 92UG046 and MACS1-spln virions were intact with no significant decrease in gp120 surface protein with respect to p24 capsid content compared to the corresponding untreated virus. These studies demonstrate that SPL7013 is virucidal against HIV-1 strains that utilize the CXCR4 coreceptor but not viruses tested in this study that solely use CCR5 by a mechanism that is distinct from virion disruption or loss of gp120. In addition, the mode of action by which SPL7013 prevents infection of cells with X4 and R5X4 strains is likely to differ from R5 strains of HIV-1.     

Solubility enhancement of indomethacin with poly(amidoamine) dendrimers and targeting to inflammatory regions of arthritic rats

This work includes investigation on solubility enhancement of indomethacin (IND) in the presence of poly(amidoamine) (PAMAM) dendrimers and passive targeting of the PAMAM/IND complex so formed to the inflamed regions in an animal model. The complex formation was confirmed by infrared and (1)H nuclear magnetic resonance spectroscopy methods. Solubility of IND in aqueous G4-PAMAM followed Higuchi's A(N) curve depending on pH of the solubilizing medium. The solubility was decreased upon addition of dendrimer to the IND saturated solution at various pH, indicating aggregation behavior of the PAMAM/IND complex and conforming to the Higuchi's A(N) solubility profile. The in vitro release of IND from the PAMAM/IND complex through a cellophane membrane, from a Franz diffusion cell, showed 79 +/- 3.2% drug release in 24 h. The drug release was further retarded in the presence of human serum albumin (HSA) suggesting the significance of complex HSA binding in altering in vivo behavior of the complex. Intravenous administration of the PAMAM/IND complex formulation in rats showed a two-compartment pharmacokinetic profile. Enhanced effective IND concentrations in the inflamed regions were obtained for the prolonged time period with the PAMAM/IND complex compared to the free drug in arthritic rats indicating preferred accumulation of IND to the inflamed region. The targeting efficiency of PAMAM/IND complex was 2.29 times higher compared to free drug. In contrast to the previous investigations, two interesting findings reported here are: (a) solubility behavior of IND in G4-PAMAM dendrimer deviates from linearity with increasing concentrations of dendrimer at acidic to neutral pH values and (b) inspite of lymphatic drainage, retention of PAMAM/IND complexes occurs at the inflammatory site.     

Solubility Enhancement of Indomethacin with Poly(amidoamine) Dendrimers and Targeting to Inflammatory Regions of Arthritic Rats

This work includes investigation on solubility enhancement of indomethacin (IND) in the presence of poly(amidoamine) (PAMAM) dendrimers and passive targeting of the PAMAM/IND complex so formed to the inflamed regions in an animal model. The complex formation was confirmed by infrared and ¹H nuclear magnetic resonance spectroscopy methods. Solubility of IND in aqueous G4-PAMAM followed Higuchi's A_N curve depending on pH of the solubilizing medium. The solubility was decreased upon addition of dendrimer to the IND saturated solution at various pH, indicating aggregation behavior of the PAMAM/IND complex and conforming to the Higuchi's A_N solubility profile. The in vitro release of IND from the PAMAM/IND complex through a cellophane membrane, from a Franz diffusion cell, showed 79±3.2% drug release in 24 h. The drug release was further retarded in the presence of human serum albumin (HSA) suggesting the significance of complex HSA binding in altering in vivo behavior of the complex. Intravenous administration of the PAMAM/IND complex formulation in rats showed a two-compartment pharmacokinetic profile. Enhanced effective IND concentrations in the inflamed regions were obtained for the prolonged time period with the PAMAM/IND complex compared to the free drug in arthritic rats indicating preferred accumulation of IND to the inflamed region. The targeting efficiency of PAMAM/IND complex was 2.29 times higher compared to free drug. In contrast to the previous investigations, two interesting findings reported here are: (a) solubility behavior of IND in G4-PAMAM dendrimer deviates from linearity with increasing concentrations of dendrimer at acidic to neutral pH values and (b) inspite of lymphatic drainage, retention of PAMAM/IND complexes occurs at the inflammatory site.     

Designing a molecular delivery system within a preclinical timeframe

An understanding of solid-state chemistry, including polymorphism, can reduce the time to filing an investigative new drug (IND) application. Obtaining a stable formulation for IND studies is crucial and must be the focus of much of the early solid-state chemistry research. Simple formulations such as a chemical substance in capsule (CIC)--the chemical substance can be crystalline or amorphous--are preferable for the IND trial and the solubility/dissolution rate has an important role in manufacturing IND clinical supplies. Two fast-to-IND flowcharts are presented here for exploratory IND and conventional IND. The utilization of quality by design and process analytical chemistry (PAT) concepts at an early stage will lay the foundation for the accelerated development of the medicines that are successful in the IND trials.     

Spectrophotometric determination of indinavir in bulk and pharmaceutical formulations using bromocresol purple and bromothymol blue

A simple, sensitive and selective method for the determination of indinavir (IND) in bulk and in pharmaceutical formulations is described. The method is based on extraction of this drug into chloroform as ion-pair with sulphonphthalein dyes as bromocresol purple (BCP) and bromothymol blue (BTB). The optimum conditions of the reactions were studied and optimized. The absorbance of the yellow products was measured at 427 nm for IND-BCP and 420 nm for IND-BTB. The calibration graphs were linear over the range 4.0-60.2 microg x ml(-1) of drug in chloroform, using the two dyes. The composition of the ion-pairs was established by the molar ratio method. For IND the molar ratio was determined to be 1:1 by measurement of first derivative signals at 273 nm. A calibration graph was established for 3.0-70.6 microg x ml(-1) of IND for first derivative spectrophotometry. The developed method was applied successfully for the determination of IND in pharmaceutical formulations. The data obtained were compared the data given by first derivative spectrophotometry. No differences were found.     

In vitro drug liberation and kinetics of sustained release indomethacin suppository

The aim of this study was to formulate sustained release (SR) suppositories containing indomethacin (IND) microspheres. In the first part of the study, IND microspheres were prepared by solvent evaporation method. Ethyl cellulose was used as polymer. Shape and surface characteristics, particle size and size distribution of microspheres were determined. The effect of drug: polymer ratio and stirring rate on microsphere formation, average particle size, drug loading capacity and in vitro IND release were investigated. The highest drug loading capacity was found with 1:1 drug-polymer ratio. Stirring rate caused insignificant effect on drug loading capacity but particle size. Increase in stirring rate resulted in a decrease in particle size. In the second part, SR suppositories were formulated by incorporating IND microspheres having the highest drug loaded. The bases used were PEG mixtures (400:1500:4000) and Witepsol H15. Qualitative controls and IND assay on the suppositories were carried out. The drugs released were evaluated by in vitro dissolution tests. Comparative results of SR suppositories containing IND microspheres with that of conventional ones showed that the former has sustained effect up to 480 min in vitro. Release results were evaluated kinetically and the data was fitted (Bt)(a) kinetics.     

Developmental toxicity of low generation PAMAM dendrimers in zebrafish

Biological molecules and intracellular structures operate at the nanoscale; therefore, development of nanomedicines shows great promise for the treatment of disease by using targeted drug delivery and gene therapies. PAMAM dendrimers, which are highly branched polymers with low polydispersity and high functionality, provide an ideal architecture for construction of effective drug carriers, gene transfer devices and imaging of biological systems. For example, dendrimers bioconjugated with selective ligands such as Arg-Gly-Asp (RGD) would theoretically target cells that contain integrin receptors and show potential for use as drug delivery devices. While RGD-conjugated dendrimers are generally considered not to be cytotoxic, there currently exists little information on the risks that such materials pose to human health. In an effort to compliment and extend the knowledge gleaned from cell culture assays, we have used the zebrafish embryo as a rapid, medium throughput, cost-effective whole-animal model to provide a more comprehensive and predictive developmental toxicity screen for nanomaterials such as PAMAM dendrimers. Using the zebrafish embryo, we have assessed the developmental toxicity of low generation (G3.5 and G4) PAMAM dendrimers, as well as RGD-conjugated forms for comparison. Our results demonstrate that G4 dendrimers, which have amino functional groups, are toxic and attenuate growth and development of zebrafish embryos at sublethal concentrations; however, G3.5 dendrimers, with carboxylic acid terminal functional groups, are not toxic to zebrafish embryos. Furthermore, RGD-conjugated G4 dendrimers are less potent in causing embryo toxicity than G4 dendrimers. RGD-conjugated G3.5 dendrimers do not elicit toxicity at the highest concentrations tested and warrant further study for use as a drug delivery device.     

Molecular imaging with SPECT as a tool for drug development

Molecular imaging techniques are increasingly being used as valuable tools in the drug development process. Radionuclide-based imaging modalities such as single-photon emission computed tomography (SPECT) and positron emission tomography (PET) have proven to be useful in phases ranging from preclinical development to the initial stages of clinical testing. The high sensitivity of these imaging modalities makes them particularly suited for exploratory investigational new drug (IND) studies as they have the potential to characterize in vivo pharmacokinetics and biodistribution of the compounds using only a fraction of the intended therapeutic dose (microdosing). This information obtained at an early stage of clinical testing results in a better selection among promising drug candidates, thereby increasing the success rate of agents entering clinical trials and the overall efficiency of the process. In this article, we will review the potential applications of SPECT imaging in the drug development process with an emphasis on its applications in exploratory IND studies.     

Influence of different generations of poly(propylene imine) dendrimers on human erythrocytes

The unique characteristics of dendrimers make them attractive candidates as drug carriers. However, the toxicity associated with dendrimers is a basic hurdle in their biomedical application. To ensure development of dendrimer based safe and effective delivery systems, the effect of dendrimers on human erythrocytes (RBCs) must be studied. The present study explores the toxicological behavior of different generations of poly(propylene imine) dendrimers on human RBCs. Plain fifth generation PPI dendrimers (1 mg/mL) showed approximately 6.39% hemolysis which was an indication of their suitability in drug delivery. The study was conducted on all generations from 0.5 to 5.0 G of PPI dendrimers.     

Dendrimers as biopharmaceuticals: synthesis and properties

Two general aspects which need to be considered for the successful application of dendrimers for biomedical purposes are their availability at an acceptable cost and their suitability as regards their pharmacodynamic and pharmacokinetic properties. These two aspects are covered in this review. In the first part, synthetic strategies for the preparation of dendrimers are outlined and emphasis is given to recent work on methodologies whose aim is the development of more efficient routes to dendrimers in terms of the materials used for their synthesis as well as in terms of the procedures required for their purification. These include procedures involving double-stage and double exponential synthesis, orthogonal coupling strategies, self-assembly and solid-phase approaches, as well as particularly useful synthetic protocols such as those used in "click chemistry". The second part of the review deals with the way in which the size, chemical constitution and physicochemical properties of dendrimers used for drug delivery may affect pharmacodynamic and pharmacokinetic parameters which are important considerations for drug bioavailability. This is illustrated by an overview of examples from recent work involving non-steroidal anti-inflammatory drugs, anticancer drugs and antibacterials.     

Development and optimization of thiolated dendrimer as a viable mucoadhesive excipient for the controlled drug delivery: An acyclovir model formulation

In the present study we report the development of novel thiolated dendrimers for mucoadhesive drug delivery. The thiolated dendrimers were synthesized by conjugating PAMAM dendrimer (G3.5)with cysteamine at two different molar ratios, i.e. 1:30 (DCys1) and 1:60 (DCys2). The thiolated dendrimers were further encapsulated with acyclovir (DCys1Ac and DCys2Ac) and the conjugates were characterized for thiol content, drug loading, drug release, and mucoadhesive behavior. The thiolated dendrimer conjugates showed thiol content of 10.56±0.34 and 68.21±1.84 μM/mg of the conjugate for DCys1 and DCys2, respectively. The acyclovir loading was observed to be highest in dendrimer drug conjugate (DAc) compared to other DCys1Ac and DCys2Ac conjugates. The thiolated dendrimers showed sustained release of acyclovir and showed higher mucoadhesion. The in vitro mucoadhesive activity of DCys2Ac was 1.53 and 2.89 fold higher mucoadhesion compared to DCys1Ac and DAc, respectively. These results demonstrated the usefulness of thiolated dendrimers as a mucoadhesive carrier and represent a novel platform for drug delivery.From the Clinical EditorThis study demonstrates the utility of thiolated dendrimers as mucoadhesive carriers as reported in an acyclovir delivery model system.     

Solid-state properties and dissolution behaviour of tablets containing co-amorphous indomethacin–arginine

Co-amorphous drug formulations provide the possibility to stabilize a drug in its amorphous form by interactions with low molecular weight compounds, e.g. amino acids. Recent studies have shown the feasibility of spray drying as a technique to manufacture co-amorphous indomethacin–arginine in a larger production scale. In this work, a tablet formulation was developed for a co-amorphous salt, namely spray dried indomethacin–arginine (SD IND–ARG). The effects of compaction pressure on tablet properties, physical stability and dissolution profiles under non-sink conditions were examined. Dissolution profiles of tablets with SD IND–ARG (TAB SD IND–ARG) were compared to those of tablets containing a physical mixture of crystalline IND and ARG (TAB PM IND–ARG) and to the dissolution of pure spray dried powder.Concerning tableting, the developed formulation allowed for the preparation of tablets with a broad range of compaction pressures resulting in different porosities and tensile strengths. XRPD results showed that, overall, no crystallization occurred neither during tableting nor during long-term storage. Dissolution profiles of TAB SD IND–ARG showed an immediate release of IND by erosion. The solubility of crystalline IND was exceeded by a factor of about 4, which was accompanied by a slow crystallization. For TAB PM IND–ARG, an in situ amorphization of IND in the presence of ARG was observed. As a result, a supersaturation was obtained, too, followed by a faster crystallization compared to TAB SD IND–ARG. In conclusion, the AUC_24h of TAB SD IND–ARG was twofold higher than the AUC_24h of TAB PM IND–ARG. Interestingly, different plateaus were obtained for TAB SD IND–ARG, TAB PM IND–ARG and pure SD IND–ARG after 24 h dissolution, which could be explained by the formation of different polymorphic forms of indomethacin.     

Implementation of transmission NIR as a PAT tool for monitoring drug transformation during HME processing

The aim of the work reported herein was to implement process analytical technology (PAT) tools during hot melt extrusion (HME) in order to obtain a better understanding of the relationship between HME processing parameters and the extruded formulations. For the first time two in-line NIR probes (transmission and reflectance) have been coupled with HME to monitor the extrusion of the water insoluble drug indomethacin (IND) in the presence of Soluplus (SOL) or Kollidon VA64 hydrophilic polymers. In-line extrusion monitoring of sheets, produced via a specially designed die, was conducted at various drug/polymer ratios and processing parameters. Characterisation of the extruded transparent sheets was also undertaken by using DSC, XRPD and Raman mapping. Analysis of the experimental findings revealed the production of molecular solutions where IND is homogeneously blended (ascertained by Raman mapping) in the polymer matrices, as it acts as a plasticizer for both hydrophilic polymers. PCA analysis of the recorded NIR signals showed that the screw speed used in HME affects the recorded spectra but not the homogeneity of the embedded drug in the polymer sheets. The IND/VA64 and IND/SOL extruded sheets displayed rapid dissolution rates with 80% and 30% of the IND being released, respectively within the first 20 min.     

促进我国创新药研发关键技术要素的探讨

文中通过对近年来国内化学药品研发申报的现状进行分析,结果显示我国目前是以仿制药申报为主,并逐步转向仿创结合的模式。结合在创新药研发中制药企业存在的不足,文中提出将建立创新药整体研发思路、构建适应创新研发学科需要的团队以及提高沟通交流能力作为提高我国创新药研发的关键要素。     

Dendrimers as therapeutic agents: a systematic review

Objectives Dendrimers by virtue of their therapeutic value have recently generated enormous interest among biomedical scientists. This review describes the therapeutic prospects of the dendrimer system. Key findings Their bioactivity suggests them to be promising therapeutic agents, especially in wound healing, bone mineralisation, cartilage formation and tissue repair, and in topical treatments to prevent HIV transmission. Findings also demonstrate their potential as anti‐prion, anti‐Alzheimer's, anticoagulant, antidote, anti‐inflammatory and anticancer agents. One of the dendrimer‐based formulations with activity against herpes simplex virus (VivaGel from Starpharma) has successfully completed phase I clinical trials and is expected to be available on the market soon. Summary All reports cited in this review demonstrate the use of dendrimers as medical therapeutics in different ailments. The review focuses on the current state of therapeutic potential of the dendrimer system.     

Improvement of solubility and dissolution rate of indomethacin by solid dispersions in Gelucire 50/13 and PEG4000

The aim of this study was to prepare and characterize solid dispersions of water insoluble non-steroidal anti-inflammatory drug, indomethacin (IND), with polyethylene glycol 4000 (PEG4000) and Gelucire 50/13 (Gelu.) for enhancing the dissolution rate of the drug. The solid dispersions (SDs) were prepared by hot melting method at 1:1, 1:2 and 1:4 drug to polymer ratios. Scanning electron microscopy (SEM), X-ray powder diffractometry (XRD) and differential scanning calorimetry (DSC) were used to examine the physical state of the drug. Furthermore, the solubility and the dissolution rate of the drug in its different systems were explored. The data from the XRD showed that the drug was still detectable in its solid state in all SDs of IND–Gelu. and disappeared in case of higher ratio of IND–PEG4000. DSC thermograms showed the significant change in melting peak of the IND when prepared as SDs suggesting the change in crystallinity of IND. The highest ratio of the polymer (1:4) enhanced the drug solubility about 4-folds or 3.5-folds in case of SDs of IND–PEG or IND–Gelu., respectively. An increased dissolution rate of IND at pH 1.2 and 7.4 was observed when the drug was dispersed in these carriers in form of physical mixtures (PMs) or SDs. IND released faster from the SDs than from the pure crystalline drug or the PMs. The dissolution rate of IND from its PMs or SDs increased with an increasing amount of polymer.     

Enhanced kinetic solubility profiles of indomethacin amorphous solid dispersions in poly(2-hydroxyethyl methacrylate) hydrogels

The feasibility of forming solid molecular dispersions of poorly water-soluble drugs in crosslinked poly(2-hydroethyl methacrylate) (PHEMA) hydrogel has recently been reported by our group. The purpose of the present study is to investigate the extent of enhancement of kinetic solubility of amorphous solid dispersions (ASDs) of indomethacin (IND) in crosslinked PHEMA hydrogels as compared with those based on conventional water-soluble polymer carriers. Our results show that under non-sink conditions, the initial solubility enhancement is higher for ASDs based on polyvinylpyrrolidone (PVP) and hydroxypropylmethylcellulose acetate succinate (HMPCAS), but the ability to maintain this solubility enhancement at longer times is better for ASDs based on PHEMA over a period of 24h with the extent of solubility enhancement of IND ASDs in PHEMA falling between those in PVP and HPMCAS at 10.0% IND loading after 6h and outperforming those in PVP and HPMCAS at 32.9% IND loading after 8h. The observed kinetic solubility profiles reflect the fact that the amorphous IND is released from PHEMA by a different mechanism than those from water-soluble polymer carriers. In this case, the dissolution of IND ASD from water-soluble PVP and HPMCAS is almost instantaneous, resulting in an initial surge of IND concentration followed by a sharp decline due to the nucleation and crystallization events triggered by the rapid build-up of drug supersaturation. On the other hand, the dissolution of IND ASD from insoluble crosslinked PHEMA hydrogel beads is less rapid as it is regulated by a feedback-controlled diffusion mechanism, thus avoiding a sudden surge of supersaturation in the dissolution medium. The absence of an apparent decline in drug concentration during dissolution from IND-PHEMA ASD further reflects the diminished nucleation and crystallization events during IND dissolution from hydrogel-based solid molecular dispersions. Based on the XRD analyses, a threshold IND loading level of about 34% in PHEMA has been identified, above which amorphous to crystalline transition tends to occur. Also, by selecting the appropriate particle sizes, immediate to controlled release of IND from IND-PHEMA ASD can be readily achieved as the release rate increases with decreasing PHEMA bead size. Furthermore, a robust physical stability has been demonstrated in IND-PHEMA ASD with no drug precipitation for up to 8 months at IND loadings below 16.7% under direct open cup exposure to accelerated stability conditions (40°C/75% RH).     

Alginate encapsulated mesoporous silica nanospheres as a sustained drug delivery system for the poorly water-soluble drug indomethacin

We applied a combination of inorganic mesoporous silica material, frequently used as drug carriers, and a natural organic polymer alginate (ALG), to establish a sustained drug delivery system for the poorly water-soluble drug Indomethacin (IND). Mesoporous silica nanospheres (MSNs) were synthesized using an organic template method and then functionalized with aminopropyl groups through postsynthesis. After drug loading into the pores of aninopropyl functionalized MSNs (AP-MSNs), IND loaded AP-MSNs (IND-AP-MSNs) were encapsulated by ALG through the ionic interaction. The effects of surface chemical groups and ALG layer on IND release were systematically studied using scanning electron microscopy (SEM), transmission electron microscopy (TEM), nitrogen adsorption, zeta-potential analysis and TGA analysis. The surface structure and surface charge changes of the ALG encapsulated AP-MSNs (ALG-AP-MSNs) were also investigated. The results showed that sustained release of IND from the designed drug delivery system was mainly due to the blockage effect from the coated ALG. We believe that this combination will help designing oral sustained drug delivery systems for poorly water-soluble drugs.