In order to develop safe and effective topical and transdermal formulations to treat either local skin disorders or for systemic drug delivery, it is first imperative to assess skin permeation using a reliable and comprehensive analysis method. The assessment of drug permeation into/across the skin is traditionally accomplished using Franz diffusion cells with subsequent analysis by conventional chromatographic methods such as HPLC and more recently using advanced imaging techniques. In this context, time of flight-secondary ion mass spectrometry (ToF-SIMS) offers distinctive advantages in mapping drugs within skin with high sensitivity and chemical specificity without the need for fluorescent tags or radiolabels. The work in this thesis uses the combination of conventional and advanced methods to evaluate drug permeation into the skin. This approach provides complementary and detailed information regarding the permeated mass, the permeation depth and the spatial distribution and localisation of drugs within skin. As ToF-SIMS does not produce quantitative results, due to the matrix effects, a novel high throughput method was successfully developed to quantify ToF-SIMS data. This method involved the homogenisation of skin tissue followed by microarray printing of this skin homogenate with known concentrations of active pharmaceutical ingredients, specifically imiquimod and chlorhexidine. The subsequent analysis by ToF-SIMS of the resulting array allowed the generation of a calibration curve that can be used in the quantification of the unknown drug concentration in the tape strips. This work has demonstrated the potential of a method to quantify ToF-SIMS data of drugs within skin. Imiquimod is an immune modulator drug approved by the FDA for the treatment of superficial basal cell carcinoma (BCC) but not the nodular lesions. An assessment of imiquimod permeation from commercially available Aldara™ cream into ex vivo porcine skin was carried out using the complementary approach of HPLC and ToF-SIMS analysis. This work represents the most detailed assessment to date of the true extent of permeation of imiquimod from Aldara™ cream as previous studies analysed the permeation of Aldara™ cream showed a limitation in the analytical methodology employed (i.e. analysis by HPLC only). The results showed that imiquimod does permeate into the stratum corneum but is very limited in the deeper skin cell layers. In addition, the ToF-SIMS ion images of Aldara™ cream tape strips illustrated a non-uniform distribution of imiquimod within skin which may result in a decreased efficacy of the cream to uniformly treat whole BCC lesions giving rise to the likelihood of tumour recurrence. This offers previously unobserved insights about the spatial distribution of imiquimod delivered from Aldara™ cream. As other studies have reported that Aldara™ cream has some limitations in the treatment of nodular BCC lesions due to the cream’s inability to deliver imiquimod into the deeper more invasive nodular lesions, an enhancement of imiquimod permeation is thought to be useful to overcome these limitations. Therefore, an attempt to improve delivery of imiquimod into the deeper skin layers using microemulsions and microneedles was investigated. Imiquimod microemulsions were formulated, characterised and then tested for skin permeation enhancement. However, the assessment of imiquimod permeation from the formulated microemulsions alone and with microneedle pre-treatment using HPLC and ToF-SIMS demonstrated a limited ability of the microemulsions to improve delivery of imiquimod over Aldara™ cream. In contrast, Aldara™ cream with microneedle pre-treatment using a derma stamp electric pen showed improved delivery of imiquimod into the skin. This work is believed to be the first attempt to enhance imiquimod delivery using microemulsions and microneedles. Utilising the high sensitivity offered by the ToF-SIMS instrument in the analysis of individual tape strips, an in vivo and ex vivo comparison of chlorhexidine permeation into the stratum corneum was performed using commercial products currently used in hospitals within the UK for skin antisepsis. A comparison was carried out using the tape stripping technique with subsequent analysis of an individual tape strip by ToF-SIMS. The results showed that HiBiSCRUB® 4% produces a higher concentration of chlorhexidine in the upper stratum corneum layers than other products. This work demonstrated the first known application of ToF-SIMS to compare the in vivo skin permeation of commercially available chlorhexidine products and provides the foundation for the potential application of ToF-SIMS in assessing bioequivalence of topical products.

Natural products play a pivotal role in medicine especially in the cancer arena. Many drugs that are currently used in cancer chemotherapy originated from or were inspired by nature. The toxicity and growth inhibitory activity of novel extracts of five different species of Malaysian rainforest plants, which are Melodinus species, Daphniphyllum scortechinii, Ficus fistulosa, Kopsia arborea and Tabernaemontana corymbosa, were investigated. The preliminary screening and chemical structure of jerantinine B highlighted this compound to be selected as a focus of this PhD study. Jerantinine B (JB) is one of seven novel Aspidosperma indole alkaloids isolated from the leaf extract of Tabernaemontana corymbosa. JB was previously evaluated to be one of the most potent cytotoxic jerantinine among all jerantinines against vincristine-sensitive and vincristine-resistant human oral epidermoid carcinoma cell lines. Furthermore, structural similarity of JB with the lower half of the vincristine chemical structure is therefore highlighted this compound to be a valuable candidate for biological evaluation. Herein, detailed biological evaluation of JB and its acetate derivative (jerantinine B acetate) on various human-derived carcinoma cell lines are reported. Our preliminary investigation showed significant inhibition of cell growth in sensitive and vincristine-resistant cancer cell lines, accompanied by significant inhibition of cell counts and colony formation associated with induction of apoptosis in human cancer cell lines after exposure to jerantinine B. Significant dose-dependent upregulation of apoptosis biomarkers (cleaved PARP and caspase 3) was shown and further confirmed apoptosis. Inhibition of cancer cell migration and invasion was observed after exposure of cells to JB. Profound G2/M cell cycle arrest was observed after treatment of cancer cell lines with JB. Tubulin polymerisation was significantly inhibited by JB and JB acetate. Morphological characterisations of mitotic arrest and apoptosis including microtubule disruption, multi-nucleation, DNA fragmentation, and membrane blebbing were obviously demonstrated by confocal microscopy in JB-treated cells. Indeed, significant interference in the dynamicity of microtubules caused by JB was observed and was relatively similar to that caused by vincristine and colchicine. Binding affinity of JB to heterodimeric tubulin protein was confirmed, measured and compared to vincristine and colchicine. Both JB and colchicine were shown to exhibit a cooperative binding response compared to vincristine which was characterised by no cooperative interaction with tubulin protein. The high-resolution crystal structure was obtained finally showing that JB acetate binds to the colchicine site on microtubules. Polo-like kinase 1 (PLK-1; an early trigger for the G2/M transition) was also dose-dependently inhibited by JB. Investigating a secondary mechanism by which jerantinine induces apoptosis, inhibits microtubule assembly and overcomes vincristine resistance via production of reactive oxygen species was considered. JB induced significant levels of ROS in treated cancer cells including vincristine-resistant cells, possibly contributing to their growth inhibitory and apoptotic destiny. Vincristine was unable to induce reactive oxygen production in vincristine-resistant cells. JB acetate demonstrated enhanced chemical stability compared to JB which could be the reason behind the greater potency of jerantinine B acetate compared to JB. Improvement in the targeting JB acetate to HER2-overexpressing breast cancer cells was attempted following conjugation of JB acetate to the HER2 affibody; potency was enhanced 2.25-fold when cells were exposed to the conjugate compared with JB acetate alone. The jerantinine alkaloid family represent a promising class of novel alkaloids which may produce putative clinical candidate molecules with broad-spectrum antitumour activity.

Cardiovascular diseases (CVD) is a leading cause of death in the world. Despite effective treatment regimens for ischaemic heart disease (IHD) and ischaemic stroke, mortality and recurrence rates remain high. Antiplatelet therapy is on effective treatment and reduces the risk of recurrent heart attack and stroke. Nevertheless, there are patients who stopped or interrupted their antiplatelet therapy for certain reasons or some patients may be resistant or poor responders to antiplatelet therapy. Furthermore, there is evidence of rebound effect in platelet activity after antiplatelet cessation and this may associate with increased risk of cardiovascular event. This thesis is divided into five main chapters (chapters 3 to 7) which attempt to provide data to help resolve the uncertainty. Chapter 1 highlights the background of cardiovascular diseases and the global burden of cardiovascular and cerebrovascular diseases. The metabolism of platelets, antiplatelet therapy and current antiplatelet therapy guidelines are described, followed by discussion of the risk of cardiovascular event and changes in antiplatelet therapy. Chapter 2 describes the data source from Virtual International Stroke Trial Archive (VISTA) and National Health Service Greater Glasgow and Clyde (NHSGGC) Safe Haven, followed by definition of outcome measures. In chapter 3, Virtual International Stroke Trial Archive (VISTA) data was examined to test whether continue with the same antiplatelet therapy or changing to a new antiplatelet regimen reduces the risk of subsequent events in patients who experience a stroke whilst taking antiplatelet therapy. The findings indicate that subjects who switch to a new antiplatelet regimen after stroke did not have a lower early recurrence rate than subjects who continued with the same antiplatelet therapy. Observations on bleeding complications were similar in both groups. However, changing antiplatelet regimen after stroke was associated with more favourable functional outcome across a full scale modified Rankin Scale (mRS) at 90 days. In chapter 4, association between early or later initiation of antiplatelet with a recurrent ischaemic stroke and bleeding complications was assessed using VISTA data. The findings indicate that there was no association between a recurrent ischaemic stroke and timing of initiation of antiplatelet drug after stroke. However, early initiation was associated with increased risk of bleeding. In terms of functional outcomes, this study demonstrated that the mid-time and late initiation of antiplatelet therapy after acute stroke are associated with better functional outcomes compared with early initiation. In chapter 5, a nested case-control study was performed to explore the rate of antiplatelet cessation and interruption in a sample of patients with recent ischaemic stroke and to assess the risk of cardiovascular events associated with cessation and interruption of antiplatelet. It was found that there was no increased risk of cardiovascular event among patients who had early cessation or interrupted/stopped antiplatelet therapy within 90 days following acute ischaemic stroke. In chapter 6, the incidence and predictors of cardiovascular events after DAPT cessation were evaluated. The incidence of cardiovascular event while taking DAPT and following discontinuation of DAPT was 15.7% and 16.7% respectively. This study found that increasing age was associated with an increased risk of cardiovascular event, whereas, revascularization-treated patients and longer duration of DAPT, were each associated with a decreased risk. The duration of DAPT six months and less was associated a significantly higher risk for cardiovascular event. In chapter 7, an untargeted metabolomics analysis was performed while on DAPT (aspirin plus ticagrelor) and once they stopped ticagrelor to identify metabolite changes associated with cardiovascular events after stopping DAPT. Ten ACS patients were recruited in this study and data were analysed for seven patients. Three hundred eleven putative metabolites were identified. This study found 16 putative metabolites significantly altered following ticagrelor cessation. Of these, seven metabolites were from lipid pathway and down-regulated some up to 3-fold. On the other hand, adenosine, from nucleotide metabolism was upregulated up to 2.6-fold. It concluded that there are changes in numerous pathways following DAPT discontinuation and whether these changes differ in patients who have cardiovascular event after stopping DAPT warrant further investigation. In chapter 8, a summary of the findings of this thesis are presented as well as the future directions of research in this area.

The decision whether or not to discharge an outpatient is vital in determining outpatient clinic attendance numbers, directly affecting overall patient care efficiency. A review of the factors influencing discharge decisions revealed that there was limited evidence of these factors and a lack of understanding how clinicians take discharge decisions. This project’s objectives were to describe the influential factors on discharge decisions from the clinicians’ and patients’ perspectives, to demonstrate the development and clinical evaluation of a novel "Traffic-light” design dermatology outpatient discharge information checklist to improve appropriateness and consistency in discharge decision-making. Semi-structured interviews were carried out with 40 consultant dermatologists across England. 148 influences were generated and thematically analysed manually and using NVivo10 software. A wide array of nonclinical factors, clinician-based, patient-based, practice-based and policy-based, influence discharge decision-making. Observations of 64 consultations and 56 semi-structured interviews with dermatology outpatients were carried out to understand their experience concerning the decision for their discharge. Twelve of 31 patients (39%) who were discharged considered their discharge inappropriate. A three-round Delphi exercise with 17 dermatology consultants (100% response) was carried out to reach agreement on what a high quality discharge information checklist should contain. There was strong inter-rater reliability (ICC=0.958) and fair inter-rater agreement (Fleiss Kappa=0.269). Thirteen items were identified that formed the "Trafficlight" design checklist. Twelve (67%) dermatology clinicians who evaluated the checklist found it useful. This study has demonstrated the importance of approaching discharge decision taking in an informed, structured manner. The checklist provide the basis for making discharge decisions more systematic, auditable and transparent, improving patient safety and optimising healthcare costs. These methods are potentially useful in other clinical disciplines.

Gelation of polysaccharides under shear conditions results in the formation of a weak gel which is able to resist elastic mechanical deformation at small strains but will flow if subjected to higher strains. The resulting material, described in the literature as a fluid gel or a sheared gel, consists of gelled microparticles which can be formulated to collectively act in bulk, as pourable viscoelastic fluids whilst retaining true gel characteristics at the micro/nano level. The tuneable behaviour of these fluid gel systems makes them potentially useful in pharmaceutical applications. Fluid gels prepared from gellan gum are particularly attractive, due to its sensitivity to physiological fluids, unique rheological and physical properties, and current regulatory approval for use as a food additive and pharmaceutical excipient. Therefore, the aim of the present study was to investigate gellan gum fluid gels as a new modified release drug delivery platform. The formation and production of fluid gels using low acyl (LA) gellan, high acyl (HA) gellan and LA HA gellan blends was investigated and applied in three different dosage forms; a modified release oral liquid, a mucoadhesive nasal spray and a topical formulation. A modified release oral liquid was designed using a fluid gel prepared from LA gellan gum. It was demonstrated that 0.75 % w/w LA gellan gum fluid gel, containing ibuprofen as the drug, could be formulated to have a similar viscosity profile as a marketed oral ibuprofen liquid. Furthermore, due to the acid insolubility of gels prepared from LA gellan, no ibuprofen was released in stimulated gastric fluid. Subsequent release at pH 7.4 however, was affected by the duration of exposure and strength of the acidic pH used and a linear relationship between onset of release and the preceding duration of acid exposure was observed. Delayed release was a result of increasing gel stiffness, a consequence of the acidity of the initial release media and exposure time. A much faster release rate was measured when exposure time in acid was 10 min compared with 60 min. This study highlights the potential to design fluid gels that are tuned to have a specified stiffness at a particular pH and exposure time allowing the intelligent design oral liquids with specific modified release behaviour. The second part of this study was to prepare mucoadhesive nasal drug delivery systems to enhance the retention of the nasal spray dosage form in the nasal cavity. Several groups have investigated using LA gellan solution as a drug delivery vehicle but only limited research however, has been performed on HA gellan for this purpose, despite its properties being more conducive to mucoadhesion. HA gellan (even with low concentration 0.25 % w/w) produces highly elastic gels below 60 °C which make it difficult to spray using a mechanical spray device. To address this problem, fluid gels were prepared as these systems can behave as sprayable viscoelastic fluids. In this study the rheological behaviour was investigated and the mucoadhesion behaviour of fluid gels prepared from the two different types of gellan (HA and LA) and fluid gels prepared from a blend of LA HA gellan. The results demonstrated that by preparing fluid gels from a blend of LA HA gellan, the rheological properties were sufficient to spray through a standard nasal spray device. Moreover, the fluid gels significantly enhanced both HA and LA gellan mucoadhesion properties. In the final part of this thesis the topical application of gellan fluid gels was explored. A range of gellan fluid gel formulations were prepared containing diclofenac sodium for topical application. The rheological results showed that it was possible to produce a topical formulation with a viscosity and the mechanical strength similar to that of the commercially available Voltaren® gel using 1 % w/w of a 50:50 LA HA gellan blend. The permeation results highlighted that the penetration of diclofenac through procaine tissue is significantly increased by increasing gellan concentration and decreasing sodium ion concentration in the formulation.

Silicon makes up almost 28% of the Earth's crust and within that crust, quartz (crystalline silica) is one of the most abundant minerals. Exposure to quartz can occur in a number of occupations, including the mining and construction industries in which respirable quartz particles are generated and become airborne. Inhalation of quartz can lead to the fibrosing lung disease silicosis and cancer. Silicosis has been recognised for many decades as one of the most prevalent occupational lung diseases. In 1997, an IARC working Group classified quartz as a class 1 lung carcinogen, but only in some industries, suggesting that the quartz hazard is a variable entity. The reactivity of the quartz surface may underlie its ability to cause inflammation and treatments that ameliorate this reactivity would then reduce the quartz hazard. In the present study the effect of treating quartz with aluminium lactate, a procedure reported to decrease the quartz hazard, on the highly reactive quartz surface and on proinflammatory events in the rat lung were explored. Aluminium lactate-treated quartz showed a reduced surface reactivity as measured by electron spin resonance. Eighteen hours post-instillation of quartz into the rat lung, there was massive inflammation as indicated by the number of neutrophils in the bronchoalveolar lavage (BAL) and an increase in BAL macrophage inflammatory protein-2 (MIP-2). However, aluminium lactate-treated quartz had no significant effect when compared to control. Epithelial damage as indicated by BAL protein and gamma glutamyl transpeptidasea lso increased with quartz instillation but not with aluminium lactate-treated quartz and furthermore, quartz induced an increase in MIP-2 mRNA content of BAL cells while aluminium lactate-treated quartz had no effect compared to controls. There was an increase in nuclear binding of the transcription factor nuclear factor-kappa B (NF-xB) in the quartz exposed BAL cells and again, no effect on nuclear NF-xB binding in BAL cells from aluminium lactate-treated quartz instilled rats. In addition, the effect of aluminium lactate and PVNO quartz treatment on DNA damage, cell cytotoxicity and particle uptake by A549 cells was assessed. DNA strand breakage, as produced by quartz at non-toxic concentrations, could be completely prevented by both coating materials. Particle uptake by A549 cells appeared to be significantly inhibited by the PVNO coating, and to a lesser extent by the aluminium lactate coating, demonstrating that respirable quartz particles induce oxidative DNA damage in human lung epithelial cells and indicating that the surface properties of the quartz as well as particle uptake by these target cells are important in the cytotoxic and genotoxic effects of quartz in vitro. Finally, the role played by surface area and specific reactivity in the acute inflammatory response to particles was investigated. Acute inflammatory response following instillation of particles has been used to evaluate hazard but has been criticised because of the non-physiological delivery and the problems of local overload. Here, a number of low toxicity dusts of various particle sizes were instilled and the neutrophil influx into the lung 18-24 hours post-instillation assessed. The extent of inflammation was shown to be a function of the surface area instilled and ultrafine particles, which present a case of high surface area per unit mass, were inflammogenic pro rata with their surface area. There is no evidence that ultrafine particles of carbon black, titanium dioxide or polystyrene have any special reactivity in addition to their large surface area. We further tested whether this approach could be used to model the reactivity of highly toxic dusts. Rats were instilled with either quartz or aluminium lactate-treated quartz and, as anticipated, the high specific surface reactivity of quartz meant that it was much more inflammogenic than was predicted using the relationship described for `low toxicity' dusts. This approach represents the possibility of modelling potential toxicity for nuisance dusts based on the inflammatory response of a given instilled surface area dose.

No description available.

The G protein-coupled receptor FFA2 is a key mediator of short chain fatty acid signalling, which are produced in the gut via fermentation of poorly digested carbohydrates by the gut microbiota. Therefore, FFA2 has attracted interest as a potential therapeutic target for metabolic and inflammatory diseases. However, several limitations have hindered validation of FFA2 as a drug target, including the limited understanding of the molecular determinants of ligand binding and species-specific differences in pharmacology. Herein, novel tool compounds and assay systems were developed for FFA2 and utilised to address some of these limitations. Following the characterisation of functional assays for detection of FFA2 signalling, these were employed to examine the structure-activity relationship and pharmacology of FFA2 agonists versus antagonists. To assess how the pharmacology of FFA2 ligands is defined by their mode of binding, a radioligand binding assay was developed using a tritiated form of FFA2 antagonist GLPG0974 that was utilised in combination with site-directed mutagenesis and homology modelling to explore FFA2 ligand binding sites. These studies showed that FFA2 agonist binding was defined by an essential interaction between the ligand carboxylate and an orthosteric Arg-His-Arg triad. In contrast, FFA2 antagonists only required one orthosteric arginine for high-affinity binding and could tolerate modifications of the carboxylate moiety. This knowledge was applied to develop an antagonist-based fluorescent tracer for FFA2 that was utilised in BRET binding assays but displayed complex pharmacological behaviour that was shown to be based on the bitopic nature of FFA2 antagonists. The secondary binding site of FFA2 antagonists was also related to their lack of action at rodent orthologues of FFA2, whose molecular basis was explored using homology models of human and murine FFA2. This facilitated the identification of a single lysine to arginine variation at position 2.60 that might provide a basis for antagonist selectivity. Extending these studies to agonist function demonstrated that removal of the positive charge at this position produced a signalling-biased form of FFA2, in which only coupling to Gi G proteins was fully maintained. In summary, these findings contribute to understanding the complex pharmacology of FFA2 ligands and the underlying mechanisms that define their function, and conclusions drawn from these studies may help advance future efforts to validate the therapeutic potential of targeting FFA2.

Background: Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the UK and has a poor 60% 5-year survival rate. The Wnt signalling pathway is fundamental for homeostasis of the intestinal epithelium and its deregulation drives development of CRC and induces DNA damage. Histone-2AX (H2AX) is a component of the nucleosome whose phosphorylated form, γH2AX, is a marker of DNA damage. Objectives: Using a well-characterised inducible CRC mouse model of early Wnt deregulation, and established Apc-deficient driven tumour and ex vivo organoid models, we have assessed whether the spontaneous DNA damage generated in these models can be targeted using 111In-anti-γH2AX-TAT (RH2AX), a radio-labelled antibody targeting γH2AX. Methods: Deletion of the Apc gene was effected in the intestine of VilCreERApcfl/fl and Lgr5CreERApcfl/fl models by intraperitoneal or oral induction with tamoxifen. γH2AX immunohistochemical (IHC) characterisation of intestines were performed as well as γH2AX whole mount immunofluorescent analysis on organoids derived from them. RH2AX, an anti-γH2AX antibody conjugated to the cell-penetrating peptide TAT to allow cellular internalisation and nuclear localisation, was used in these models as an imaging agent SPECT/CT imaging and biodistribution studies were conducted after oral induction of VilCreERApcfl/fl and intravenous injection of RH2AX. γH2AX and Lgr5 FACS analysis were carried out on intestinal crypt cells of VilCreERApcfl/fl mice expressing Lgr5-EGFP reporter. Results: Intestinal Apc deficiency increased DNA damage levels in the small intestine of both dysplastic (VilCreERApcfl/fl) and tumour CRC mouse (Lgr5CreERApcfl/fl) models. Apc-deficiency-associated DNA damage is most likely generated through WNT signalling pathway activation and, more specifically, by c-Myc transcription. For the first time, we demonstrated that intestinal dysplasia can be identified through in vivo SPECT imaging, using low SA RH2AX treatment. Low SA RIC treatment in intestinal dysplasia increased the DNA damage levels in healthy and Apc-deficient small and large intestines, increased proliferation in the Apc-deficient tissue and resulted in variable levels of apoptosis depending on the tissue. Conclusion: These findings together indicate that DNA damage is induced by Apc-deficiency, and that there is the possibility to exploit the endogenously-increased DNA damage signal, γH2AX, to attract the RH2AX for in vivo imaging of intestinal dysplasia. This could help diagnose early stages of CRC to provide patients with the appropriate treatment sooner and increase their survival.

Parenteral nutrition (PN) provides intravenous nutritional support to patients with reduced gastrointestinal function. A PN bag comprises the basic building blocks of the food groups: lipids, glucose, amino acids, vitamins, electrolytes and trace elements. Recently there has been an increase in demand for extended storage periods for PN bags, to ease management of an increasing home care market. Prior to a PN formulation being deemed safe for a patient, a laboratory simulation is carried out on the proposed admixture under the requested storage and administration conditions. Currently only the physical stability is assessed; physical testing provides no information on the quantity of each component remaining in the bag after storage. Consequently, there is a need for assessing the chemical stability of PN to indicate the quantity of each component that remains in the PN bag. A commonly used amino acid product, Aminoven® 25, contains 16 amino acids; this work aimed to develop a HPLC assay capable of quantifying the amino acids in an aqueous PN bag containing Aminoven® 25. Fluorescence detection was used as it is a highly selective method of detection, which was preferable due to the number of components in PN. To detect the amino acids, as they don’t naturally fluoresce, derivatization was carried out using ortho-phthalaldehyde to form a fluorescing derivative. The developed assay resulted in validation of thirteen of the amino acids in Aminoven® 25. In addition, the method was shown to be unaffected by the iv presence of aqueous PN components, so this method is suitable for quantifying thirteen amino acids in aqueous PN containing Aminoven® 25. This assay can be used for assessing the stability during stability testing and confirming the quantity of amino acids after compounding for quality control release.