Photodynamic therapy with verteporfin for juxtafoveal choroidal neovascularisation secondary to pathological myopia

The treatment of choroidal neovascularisation (CNV) secondary to pathological myopia has presented a number of problems to ophthalmologists over the years, but the advent of photodynamic therapy (PDT) with verteporfin has changed how we manage these patients. Until PDT became available, the use of laser photocoagulation for extra and juxtafoveal lesions had been shown to be effective in the short term in preventing loss of vision, although the risk of regrowth of CNV and undertreatment were well recognised. However, even in apparent successful cases of photocoagulation, laser scar enlargement and creepage into the fovea in the mid-to-long term often occurred with resulting loss of central vision.1 Other options for treatment were very limited with little evidence that other modalities such as transpupillary thermotherapy or submacular surgery and macular transplantation surgery would be successful in highly myopic eyes. The evidence for the role of PDT and verteporfin CNV secondary to pathological myopia comes from the verteporfin in photodynamic therapy (VIP) study that has shown how effective this treatment is in eyes with subfoveal CNV.2, 3 Now in this publication, Lam et al4 from Hong Kong have shown that PDT is also effective in juxtafoveal CNV, with high myopia. They performed a small prospective study of 11 patients of mean age 44.8 years, with 12 months of follow-up. They found that there was a mean improvement of 1.8 lines of LogMAR best-corrected visual acuity (BCVA) at 12 months, with a mean number of 2.3 PDT treatments. The most rapid improvement occurred within the first 3 months of treatment and by 12 months none of the patients had suffered a deterioration in BCVA from baseline. There were no cases of adverse effects from the infusion or laser treatment. For ophthalmologists dealing with patients with CNV secondary to causes other than AMD, this is further evidence of the effectiveness of PDT with verteporfin in maintaining vision. These patients are likely to be younger than those with AMD and are likely to be in active employment and supporting families, and clearly the preservation of best vision possible is imperative in this group. It is therefore encouraging for ophthalmologists in the United Kingdom that the verteporfin in PDT Cohort Study (VPDT Study) includes the ability to treat patients with subfoveal CNV secondary to high myopia if they fulfill National Institute of Clinical Excellence guidelines, and will allow representations to be made on an individual basis for treatment of juxtafoveal lesions.5 For those ophthalmologists used to juggling increased patient expectations with scarce NHS resources, this is promising news and will allow us to offer a better standard of care to our patients.

Verteporfin photodynamic therapy cohort study:report 1: effectiveness and factors influencing outcomes

Purpose - To compare the visual outcomes after verteporfin photodynamic therapy (VPDT) administered in routine clinical practice with those observed in the Treatment of Age-related macular degeneration with Photodynamic therapy (TAP) trials and to quantify the effects of clinically important baseline covariates on outcome. Design - A prospective longitudinal study of patients treated with VPDT in 45 ophthalmology departments in the United Kingdom with expertise in the management of neovascular age-related macular degeneration (nAMD). Participants - Patients with wholly or predominantly classic choroidal neovascularization (CNV) of any cause with a visual acuity =20/200 in the eye to be treated. Methods - Refracted best-corrected visual acuity (BCVA) and contrast sensitivity were measured in VPDT-treated eyes at baseline and subsequent visits. Eyes were retreated at 3 months if CNV was judged to be active. Baseline angiograms were graded to quantify the percentages of classic and occult CNV. Treated eyes were categorized as eligible or ineligible for TAP, or unclassifiable. Main Outcome Measures - Best-corrected visual acuity and contrast sensitivity during 1 year of follow-up after initial treatment. Results - A total of 7748 treated patients were recruited. Data from 4043 patients with a diagnosis of nAMD were used in the present analysis. Reading center determination of lesion type showed that 87% were predominantly classic CNV. Eyes received 2.4 treatments in year 1 and 0.4 treatments in year 2. Deterioration of BCVA over 1 year was similar to that observed in the VPDT arms of the TAP trials and was not influenced by TAP eligibility classification. Best-corrected visual acuity deteriorated more quickly in current smokers; with increasing proportion of classic CNV, increasing age, and better baseline BCVA; and when the fellow eye was the better eye.

Photodynamic therapy:a treatment option in choroidal neovascularisation secondary to punctate inner choroidopathy

Punctate inner choroidopathy is an idiopathic inflammatory ocular disorder characteristically seen in young myopic women. Visual prognosis is generally good but sight threatening choroidal neovascularisation may develop in up to 40% patients.1 We discuss verteporfin photodynamic therapy in subfoveal choroidal neovascularisation secondary to punctate inner choroidopathy that failed to respond to oral corticosteroids and had poor results with submacular surgery in the contralateral eye.

Decreased efficiency of trypsinization of cells following photodynamic therapy:evaluation of a role for tissue transglutaminase

Identifying the cellular responses to photodynamic therapy (PDT) is important if the mechanisms of cellular damage are to be fully understood. The relationship between sensitizer, fluence rate and the removal of cells by trypsinization was studied using the RIF-1 cell line. Following treatment of RIF-1 cells with pyridinium zinc (II) phthalocyanine (PPC), or polyhaematoporphyrin at 10 mW cm−2 (3 J cm−2), there was a significant number of cells that were not removed by trypsin incubation compared to controls. Decreasing the fluence rate from 10 to 2.5 mW cm−2 resulted in a two-fold increase in the number of cells attached to the substratum when PPC used as sensitizer; however, with 5,10,15,20 meso-tetra(hydroxyphenyl) chlorin (m-THPC) there was no resistance to trypsinization following treatment at either fluence rate. The results indicate that resistance of cells to trypsinization following PDT is likely to be both sensitizer and fluence rate dependent. Increased activity of the enzyme tissue-transglutaminase (tTGase) was observed following PPC-PDT, but not following m-THPC-PDT. Similar results were obtained using HT29 human colonic carcinoma and ECV304 human umbilical vein endothelial cell lines. Hamster fibrosarcoma cell (Met B) clones transfected with human tTGase also exhibited resistance to trypsinization following PPC-mediated photosensitization; however, a similar degree of resistance was observed in PDT-treated control Met B cells suggesting that tTGase activity alone was not involved in this process.

Defining response to anti-VEGF therapies in neovascular AMD

The introduction of anti-vascular endothelial growth factor (anti-VEGF) has made significant impact on the reduction of the visual loss due to neovascular age-related macular degeneration (n-AMD). There are significant inter-individual differences in response to an anti-VEGF agent, made more complex by the availability of multiple anti-VEGF agents with different molecular configurations. The response to anti-VEGF therapy have been found to be dependent on a variety of factors including patient’s age, lesion characteristics, lesion duration, baseline visual acuity (VA) and the presence of particular genotype risk alleles. Furthermore, a proportion of eyes with n-AMD show a decline in acuity or morphology, despite therapy or require very frequent re-treatment. There is currently no consensus as to how to classify optimal response, or lack of it, with these therapies. There is, in particular, confusion over terms such as ‘responder status’ after treatment for n-AMD, ‘tachyphylaxis’ and ‘recalcitrant’ n-AMD. This document aims to provide a consensus on definition/categorisation of the response of n-AMD to anti-VEGF therapies and on the time points at which response to treatment should be determined. Primary response is best determined at 1 month following the last initiation dose, while maintained treatment (secondary) response is determined any time after the 4th visit. In a particular eye, secondary responses do not mirror and cannot be predicted from that in the primary phase. Morphological and functional responses to anti-VEGF treatments, do not necessarily correlate, and may be dissociated in an individual eye. Furthermore, there is a ceiling effect that can negate the currently used functional metrics such as >5 letters improvement when the baseline VA is good (ETDRS>70 letters). It is therefore important to use a combination of both the parameters in determining the response.The following are proposed definitions: optimal (good) response is defined as when there is resolution of fluid (intraretinal fluid; IRF, subretinal fluid; SRF and retinal thickening), and/or improvement of >5 letters, subject to the ceiling effect of good starting VA. Poor response is defined as <25% reduction from the baseline in the central retinal thickness (CRT), with persistent or new IRF, SRF or minimal or change in VA (that is, change in VA of 0+4 letters). Non-response is defined as an increase in fluid (IRF, SRF and CRT), or increasing haemorrhage compared with the baseline and/or loss of >5 letters compared with the baseline or best corrected vision subsequently. Poor or non-response to anti-VEGF may be due to clinical factors including suboptimal dosing than that required by a particular patient, increased dosing intervals, treatment initiation when disease is already at an advanced or chronic stage), cellular mechanisms, lesion type, genetic variation and potential tachyphylaxis); non-clinical factors including poor access to clinics or delayed appointments may also result in poor treatment outcomes. In eyes classified as good responders, treatment should be continued with the same agent when disease activity is present or reactivation occurs following temporary dose holding. In eyes that show partial response, treatment may be continued, although re-evaluation with further imaging may be required to exclude confounding factors. Where there is persistent, unchanging accumulated fluid following three consecutive injections at monthly intervals, treatment may be withheld temporarily, but recommenced with the same or alternative anti-VEGF if the fluid subsequently increases (lesion considered active). Poor or non-response to anti-VEGF treatments requires re-evaluation of diagnosis and if necessary switch to alternative therapies including other anti-VEGF agents and/or with photodynamic therapy (PDT). Idiopathic polypoidal choroidopathy may require treatment with PDT monotherapy or combination with anti-VEGF. A committee comprised of retinal specialists with experience of managing patients with n-AMD similar to that which developed the Royal College of Ophthalmologists Guidelines to Ranibizumab was assembled. Individual aspects of the guidelines were proposed by the committee lead (WMA) based on relevant reference to published evidence base following a search of Medline and circulated to all committee members for discussion before approval or modification. Each draft was modified according to feedback from committee members until unanimous approval was obtained in the final draft. A system for categorising the range of responsiveness of n-AMD lesions to anti-VEGF therapy is proposed. The proposal is based primarily on morphological criteria but functional criteria have been included. Recommendations have been made on when to consider discontinuation of therapy either because of success or futility. These guidelines should help clinical decision-making and may prevent over and/or undertreatment with anti-VEGF therapy.

The use of amino acids to enhance the aerosolisation of spray-dried powders for pulmonary gene therapy

Background Pulmonary delivery of gene therapy offers the potential for the treatment of a range of lung conditions, including cystic fibrosis, asthma and lung cancer. Spray-drying may be used to prepare dry powders for inhalation; however, aerosolisation of such powders is limited, resulting in poor lung deposition and biological functionality. In this study, we examine the use of amino acids (arginine, aspartic acid, threonine, phenylalanine) to enhance the aerosolisation of spray-dried powders containing model non-viral gene vectors. Methods Lipid/polycation/pDNA (LPD) vectors, in the presence or absence of amino acids, were dispersed in lactose solutions, and spray-dried to produce appropriately sized dry powders. Scanning electron microscopy and laser diffraction were used to determine particle morphology and diameter, respectively. Gel electrophoresis was used to examine the influence of amino acids on the structural integrity of the LPD complex. In vitro cell (A.549) transfection was used to determine the biological functionality of the dry powders, and the in vitro aerosolisation performance was assessed using a multistage liquid impinger (MSLI). Results Both gel electrophoresis and in vitro cell transfection indicated that certain amino acids (aspartic acid, threonine) can adversely affect the integrity and biological functionality of the LPD complex. All amino acids significantly increased the aerosolisation of the powder, with the arginine and phenylalanine powders showing optimal deposition in the lower stages of the MSLI. Conclusions Amino acids can be used to enhance the aerosolisation of spray-dried powders for respiratory gene delivery, allowing the development of stable and viable formulations for pulmonary gene therapy.

Purification of plasmid DNA for use in human gene therapy

Two key issues defined the focus of this research in manufacturing plasmid DNA for use In human gene therapy. First, the processing of E.coli bacterial cells to effect the separation of therapeutic plasmid DNA from cellular debris and adventitious material. Second, the affinity purification of the plasmid DNA in a Simple one-stage process. The need arises when considering the concerns that have been recently voiced by the FDA concerning the scalability and reproducibility of the current manufacturing processes in meeting the quality criteria of purity, potency, efficacy, and safety for a recombinant drug substance for use in humans. To develop a preliminary purification procedure, an EFD cross-flow micro-filtration module was assessed for its ability to effect the 20-fold concentration, 6-time diafiltration, and final clarification of the plasmid DNA from the subsequent cell lysate that is derived from a 1 liter E.coli bacterial cell culture. Historically, the employment of cross-flow filtration modules within procedures for harvesting cells from bacterial cultures have failed to reach the required standards dictated by existing continuous centrifuge technologies, frequently resulting in the rapid blinding of the membrane with bacterial cells that substantially reduces the permeate flux. By challenging the EFD module, containing six helical wound tubular membranes promoting centrifugal instabilities known as Dean vortices, with distilled water between the Dean number's of 187Dn and 818Dn,and the transmembrane pressures (TMP) of 0 to 5 psi. The data demonstrated that the fluid dynamics significantly influenced the permeation rate, displaying a maximum at 227Dn (312 Imh) and minimum at 818Dn (130 Imh) for a transmembrane pressure of 1 psi. Numerical studies indicated that the initial increase and subsequent decrease resulted from a competition between the centrifugal and viscous forces that create the Dean vortices. At Dean numbers between 187Dn and 227Dn , the forces combine constructively to increase the apparent strength and influence of the Dean vortices. However, as the Dean number in increases above 227 On the centrifugal force dominates the viscous forces, compressing the Dean vortices into the membrane walls and reducing their influence on the radial transmembrane pressure i.e. the permeate flux reduced. When investigating the action of the Dean vortices in controlling tile fouling rate of E.coli bacterial cells, it was demonstrated that the optimum cross-flow rate at which to effect the concentration of a bacterial cell culture was 579Dn and 3 psi TMP, processing in excess of 400 Imh for 20 minutes (i.e., concentrating a 1L culture to 50 ml in 10 minutes at an average of 450 Imh). The data demonstrated that there was a conflict between the Dean number at which the shear rate could control the cell fouling, and the Dean number at which tile optimum flux enhancement was found. Hence, the internal geometry of the EFD module was shown to sub-optimal for this application. At 579Dn and 3 psi TMP, the 6-fold diafiltration was shown to occupy 3.6 minutes of process time, processing at an average flux of 400 Imh. Again, at 579Dn and 3 psi TMP the clarification of the plasmid from tile resulting freeze-thaw cell lysate was achieved at 120 Iml1, passing 83% (2,5 mg) of the plasmid DNA (6,3 ng μ-1 10.8 mg of genomic DNA (∼23,00 Obp, 36 ng μ-1 ), and 7.2 mg of cellular proteins (5-100 kDa, 21.4 ngμ-1 ) into the post-EFD process stream. Hence the EFD module was shown to be effective, achieving the desired objectives in approximately 25 minutes. On the basis of its ability to intercalate into low molecular weight dsDNA present in dilute cell lysates, and be electrophoresed through agarose, the fluorophore PicoGreen was selected for the development of a suitable dsDNA assay. It was assesseel for its accuracy, and reliability, In determining the concentration and identity of DNA present in samples that were eleclrophoresed through agarose gels. The signal emitted by intercalated PicoGreen was shown to be constant and linear, and that the mobility of the PicaGreen-DNA complex was not affected by the intercalation. Concerning the secondary purification procedure, various anion-exchange membranes were assessed for their ability to capture plasmid DNA from the post-EFD process stream. For a commercially available Sartorius Sartobind Q15 membrane, the reduction in the equilibriumbinding capacity for  ctDNA in buffer of increasing ionic demonstrated that DNA was being.adsorbed by electrostatic  interactions only. However, the problems associated with fluid distribution across the membrane demonstrated that the membrane housing was the predominant cause of the .erratic breakthrough curves. Consequently, this would need to be rectified before such a membrane could be integrated into the current system, or indeed be scaled beyond laboratory scale. However, when challenged with the process material, the data showed that considerable quantities of protein (1150 μg) were adsorbed preferentially to the plasmid DNA (44 μg). This was also shown for derived Pall Gelman UltraBind US450 membranes that had been functionalised by varying molecular weight poly-L~lysine and polyethyleneimine ligands. Hence the anion-exchange membranes were shown to be ineffective in capturing plasmid DNA from the process stream. Finally, work was performed to integrate a sequence-specific DNA·binding protein into a single-stage DNA chromatography, isolating plasmid DNA from E.coli cells whilst minimising the contamination from genomic DNA and cellular protein. Preliminary work demonstrated that the fusion protein was capable of isolating pUC19 DNA into which the recognition sequence for the fusion-protein had been inserted (pTS DNA) when in the presence of the conditioned process material. Althougth the pTS recognition sequence differs from native pUC19 sequences by only 2 bp, the fusion protein was shown to act as a highly selective affinity ligand for pTS DNA alone. Subsequently, the scale of the process was scaled 25-fold and positioned directly following the EFD system. In conclusion, the integration of the EFD micro-filtration system and zinc-finger affinity purification technique resulted in the capture of approximately 1 mg of plasmid DNA was purified from 1L of E.coli  culture in a simple two stage process, resulting in the complete removal of genomic DNA and 96.7% of cellular protein in less than 1 hour of process time.

The use of absorption enhancers to enhance the despersibility of spray-dried powders for pulmonary gene therapy

Background: Pulmonary gene therapy requires aerosolisation of the gene vectors to the target region of the lower respiratory tract. Pulmonary absorption enhancers have been shown to improve the penetration of pharmaceutically active ingredients in the airway. In this study, we investigate whether certain absorption enhancers may also enhance the aerosolisation properties of spray-dried powders containing non-viral gene vectors. Methods: Spray-drying was used to prepare potentially respirable trehalose-based dry powders containing lipid-polycation-pDNA (LPD) vectors and absorption enhancers. Powder morphology and particle size were characterised using scanning electron microscopy and laser diffraction, respectively, with gel electrophoresis used to assess the structural integrity of the pDNA. The biological functionality of the powders was quantified using in vitro cell (A549) transfection. Aerosolisation from a Spinhaler® dry powder inhaler into a multistage liquid impinger (MSLI) was used to assess the in vitro dispersibility and deposition of the powders. Results: Spray-dried powder containing dimethyl-β-cyclodextrin (DMC) demonstrated substantially altered particle morphology and an optimal particle size distribution for pulmonary delivery. The inclusion of DMC did not adversely affect the structural integrity of the LPD complex and the powder displayed significantly greater transfection efficiency as compared to unmodified powder. All absorption enhancers proffered enhanced powder deposition characteristics, with the DMC-modified powder facilitating high deposition in the lower stages of the MSLI. Conclusions: Incorporation of absorption enhancers into non-viral gene therapy formulations prior to spray-drying can significantly enhance the aerosolisation properties of the resultant powder and increase biological functionality at the site of deposition in an in vitro model. Copyright © 2005 John Wiley & Sons, Ltd.