ALA and ALA hexyl ester induction of porphyrins after their systemic administration to tumour bearing mice

The use of synthetic lipophilic molecules derived from 5-aminolevulinic acid (ALA) is currently under investigation to enhance cellular ALA penetration. In this work we studied the effect of systemic administration to mice of the hexyl ester of ALA (He-ALA) on porphyrin tissue synthesis as compared to ALA. In most normal tissues as well as in tumour, He-ALA induced less porphyrin synthesis than ALA after its systemic administration either intravenous or intraperitoneal, although explant organ cultures exposed to either ALA or He-ALA revealed equally active esterases. The only tissue that accumulated higher porphyrin levels from He-ALA (seven times more than ALA) was the brain, and this correlated well with a rapid increase in ALA/He-ALA content in brain after administration of He-ALA. This may be ascribed to a differential permeability to lipophilic substances controlled by the blood–brain barrier, a feature which could be further exploited to treat brain tumours

ALA- and ALA-hexylester-induced protoporphyrin IX fluorescence and distribution in multicell tumour spheroids

Synthesis of protoporphyrin IX (PpIX) in intact murine mammary cancer cell spheroids is reported from optical sections obtained using a laser scanning confocal fluorescence microscope. EMT6 spheroids 275–350 μ m in diameter were incubated in 0.1–15 mM aminolevulinic acid (ALA) or 0.001–2 mM ALA-hexylester (h-ALA) to test the ability of both pro-drugs to diffuse into the spheroids and induce PpIX production. Spheroids incubated with ALA show significant fluorescence nonuniformity for all concentrations, with the outermost cells exhibiting greater porphyrin fluorescence. Comparable levels of fluorescence throughout the optical section are achieved with approximately 100-fold lower h-ALA concentrations, indicating that the interior cells maintain esterase activity and porphyrin synthesis and that h-ALA diffuses efficiently to the spheroid interior. Fluorescence gradients are less pronounced with h-ALA incubation, in part because of apparent saturation of esterase activity in the spheroid perimeter. Proliferating (Ki67 positive) and quiescent cell populations exhibit remarkably different h-ALA concentration dependencies. The incubation concentration resulting in maximum fluorescence with ALA is 10 mM, while the optimal concentration for h-ALA is 200-fold lower at 0.05 mM. Exceeding these optimal concentrations for both pro-drugs leads to an overall loss of fluorescence. © 2001 Cancer Research Campaign http://www.bjcancer.co

Comparative effect of ALA derivatives on protoporphyrin IX production in human and rat skin organ cultures

Samples of human and rat skin in short-term organ culture exposed to ALA or a range of hydrophobic derivatives were examined for their effect on the accumulation of protoporphyrin IX (PpIX) measured using fluorescence spectroscopy. With the exception of carbobenzoyloxy-D-phenylalanyl-5-ALA-ethyl ester the data presented indicate that, in normal tissues, ALA derivatives generate protoporphyrin IX more slowly than ALA, suggesting that they are less rapidly taken up and/or converted to free ALA. However, the resultant depot effect may lead to the enhanced accumulation of porphyrin over long exposure periods, particularly in the case of ALA-methyl ester or ALA-hexyl ester, depending on the applied concentration and the exposed tissue. Addition of the iron chelator, CP94, greatly increased PpIX accumulation in human skin exposed to ALA, ALA-methyl ester and ALA-hexyl ester. The effect in rat skin was less marked.</p

Porphyrin synthesis from ALA derivatives for photodynamic therapy. In vitro and in vivo studies

The aim of this work was to test in vitro and in vivo the efficacy of the derivatives of 5-aminolevulinic acid (ALA): hexyl-ALA (He-ALA), undecanoyl-ALA and R,S-2-(hydroximethyl)tetrahydropyranyl-ALA (THP-ALA) as pro-photosensitising agents. The compounds were assayed in a cell line derived from a murine mammary tumour, in tumour explants and after injection of the cells into mice. In vitro, undecanoyl-ALA and THP-ALA did not improve ALA efficacy in terms of porphyrin synthesis. On the other hand, half of the amount of ALA is required to obtain the same plateau amount of photosensitiser from He-ALA. However, this plateau value cannot be surpassed in spite of the four-times higher accumulation of ALA/He-ALA from the ALA derivative. This shows that He-ALA conversion to porphyrins but not He-ALA entry to the cells is limiting. Employing ionic exchange chromatography, we found that 80% of total uptake was He-ALA whereas only 20% was ALA. This suggests that the esterases, probably themselves regulated by the heme pathway, are limiting the conversion of ALA derivatives into porphyrins. A similar situation occurs with THP-ALA. Tumour explant porphyrin results correlate well with cell line data. However, i.p. injection of ALA derivatives to mice resulted in a lower porphyrin concentration in the tumour when compared to the administration of equimolar amounts of ALA, indicating that there should be retention of ALA derivatives either within the blood vessels in the initial phase of distribution and/or within the capillaries of the tumour. © 2004 Cancer Research UK.Fil: Perotti, C.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; ArgentinaFil: Fukuda, Haydee. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; ArgentinaFil: Di Venosa, Gabriela Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; ArgentinaFil: MacRobert, A.J.. No especifíca;Fil: Batlle, Alcira María del C.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; ArgentinaFil: Casas, Adriana Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; Argentin

Process Evaluation of a Dutch Community Intervention to improve Health Related Behaviour in deprived neighbourhoods

Objectives: To assess whether a community intervention on health related behaviour in deprived neighbourhoods was delivered as planned and the extent of exposure to the intervention programme. Methods: Data were gathered throughout the intervention period using minutes of meetings, registration forms and a postal questionnaire among residents in intervention and comparison neighbourhoods. Results: Overall, the intervention was delivered according to the key principles of a "community approach", although community participation could have been improved. Neighbourhood coalitions organized more than 50 health related activities in the neighbourhoods over a two-year period. Most activities were directed at attracting attention, providing information, and increasing awareness and knowledge, and at changing behaviours. Programme awareness and programme participation were 24% respectively 3% among residents in the intervention neighbourhoods. Conclusions: The process evaluation indicated that it was feasible to implement a community intervention according to the key principles of the "community approach" in deprived neighbourhoods. However, it is unlikely that the total package of intervention activities had enough strength and sufficient exposure to attain community-wide health behaviour change

Topical application of ALA and ALA hexyl ester on a subcutaneous murine mammary adenocarcinoma: tissue distribution

Although 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT) has proven to be clinically beneficial for the treatment of certain cancers, including a variety of skin cancers, optimal tissue localisation still remains a problem. An approach to improve the bioavailability of protoporphyrin IX (PpIX) is the use of ALA derivatives instead of ALA. In this work, we employed a subcutaneous murine mammary adenocarcinoma to study the tissue distribution pattern of the ALA hexyl ester (He-ALA) in comparison with ALA after their topical application in different vehicles. He-ALA induced porphyrin synthesis in the skin overlying the tumour (SOT), but it did not reach the tumour tissue as efficiently. Only 5 h after He-ALA lotion application, tumour porphyrin levels surpassed control values. He-ALA delivered in cream induced a substantially lower porphyrin synthesis in SOT, reinforcing the importance of the vehicle in the use of topical PDT. Porphyrin levels in internal organs remained almost within control values when He-ALA was employed. The addition of DMSO to ALA formulation slightly increased tumour and SOT porphyrin biosynthesis, but it did not when added to He-ALA lotion

Protoporphyrin IX enhancement by 5-aminolaevulinic acid peptide derivatives and the effect of RNA silencing on intracellular metabolism

Intracellular generation of the photosensitiser, protoporphyrin IX, from a series of dipeptide derivatives of the haem precursor, 5-aminolaevulinic acid (ALA), was investigated in transformed PAM212 murine keratinocytes, together with studies of their intracellular metabolism. Porphyrin production was substantially increased compared with equimolar ALA using N-acetyl terminated phenylalanyl, leucinyl and methionyl ALA methyl ester derivatives in the following order: Ac-L-phenylalanyl-ALA-Me, Ac-L-methionyl-ALA-Me and Ac-L-leucinyl-ALA-Me. The enhanced porphyrin production was in good correlation with improved photocytotoxicity, with no intrinsic dark toxicity apparent. However, phenylalanyl derivatives without the acetyl/acyl group at the N terminus induced significantly less porphyrin, and the replacement of the acetyl group by a benzyloxycarbonyl group resulted in no porphyrin production. Porphyrin production was reduced in the presence of class-specific protease inhibitors, namely serine protease inhibitors. Using siRNA knockdown of acylpeptide hydrolase (ACPH) protein expression, we showed the involvement of ACPH, a member of the prolyl oligopeptidase family of serine peptidases, in the hydrolytic cleavage of ALA from the peptide derivatives. In conclusion, ALA peptide derivatives are capable of delivering ALA efficiently to cells and enhancing porphyrin synthesis and photocytotoxicity; however, the N-terminus state, whether free or substituted, plays an important role in determining the biological efficacy of ALA peptide derivatives

Porphyrin accumulation induced by 5-aminolaevulinic acid esters in tumour cells growing in vitro and in vivo

The ability of 5-aminolaevulinic acid and some of its esterified derivatives to induce porphyrin accumulation has been examined in CaNT murine mammary carcinoma cells growing in culture and as tumours in vivo. Topical or intravenous administration of 5-aminolaevulinic acid-esters to mice bearing subcutaneous tumours produced lower porphyrin levels in the tumour than an equimolar dose of 5-aminolaevulinic acid. Reducing the dose of intravenous hexyl- or benzyl-ALA and topical hexyl-5-aminolaevulinic acid resulted in a dose-dependent reduction in porphyrin accumulation. A number of normal tissues accumulated higher concentrations of porphyrins than tumour tissue following intravenous administration of 5-aminolaevulinic acid-esters. Esterase activity in these normal tissues was greater than that in tumour tissue. In contrast to the situation in vivo, all of the 5-aminolaevulinic acid-esters examined were at least as effective as 5-aminolaevulinic acid when applied to cloned CaNT cells in vitro, with the drug concentration required for maximum porphyrin accumulation varying with ester chain-length. Tumour cells growing in culture released esterase activity into the medium. These findings suggest that the efficacy of 5-aminolaevulinic esters may vary depending on the esterase activity of the target tissue, and suggest caution when interpreting the findings of in vitro studies using these and similar prodrugs

Early detection of urothelial premalignant lesions using hexaminolevulinate fluorescence cystoscopy in high risk patients

<p>Abstract</p> <p>Background</p> <p>To evaluate fluorescence cystoscopy with hexaminolevulinate (HAL) in the early detection of dysplasia (DYS) and carcinoma in situ (CIS) in select high risk patients.</p> <p>Methods</p> <p>We selected 30 consecutive bladder cancer patients at high risk for progression. After endoscopic resection, all patients received (a) induction BCG schedule when needed, and (b) white light and fluorescence cystoscopy after 3 months. HAL at doses of 85 mg (GE Healthcare, Buckinghamshire, United Kingdom) dissolved in 50 ml of solvent to obtain an 8 mmol/L solution was instilled intravesically with a 12 Fr catheter into an empty bladder and left for 90 minutes. The solution was freshly prepared immediately before instillation. Cystoscopy was performed within 120 minutes of bladder emptying. Standard and fluorescence cystoscopy was performed using a double light system (Combilight PDD light source 5133, Wolf, Germany) which allowed an inspection under both white and blue light.</p> <p>Results</p> <p>The overall incidence was 43.3% dysplasia, 23.3% CIS, and 13.3% superficial transitional cell cancer. In 21 patients, HAL cystoscopy was positive with one or more fluorescent flat lesions. Of the positive cases, there were 4 CIS, 10 DYS, 2 association of CIS and DYS, 4 well-differentiated non-infiltrating bladder cancers, and 1 chronic cystitis. In 9 patients with negative HAL results, random biopsies showed 1 CIS and 1 DYS. HAL cystoscopy showed 90.1% sensitivity and 87.5% specificity with 95.2% positive predictive value and 77.8% negative predictive value.</p> <p>Conclusion</p> <p>Photodynamic diagnosis should be considered a very important tool in the diagnosis of potentially evolving flat lesions on the bladder mucosa such as DYS and CIS. Moreover, detection of dysplasic lesions that are considered precursors of CIS may play an important role in preventing disease progression. In our opinion, HAL cystoscopy should be recommended in the early follow-up of high risk patients.</p