Photodynamic diagnosis of pleural malignant lesions with a combination of 5-aminolevulinic acid and intrinsic fluorescence observation systems.

BACKGROUND: We have developed a new diagnostic method using the photosensitizer 5-aminolevulinic acid (5ALA) for diagnosing intrathoracic malignant lesions. When ingested exogenously, 5ALA is metabolized to a heme precursor, protoporphyrin IX, which stays in malignant cells and emits red to pink luminescence of about 630 nm. METHODS: We enrolled 40 patients who underwent respiratory surgery and consented to participate in this study. Twenty-eight patients had primary lung cancer, 8 metastatic lung tumors, 2 malignant pleural tumors, and 2 benign tumors. Localization of malignant lesions was attempted by observing such lesions with an autofluorescence imaging system and by comparing the color tone of the autofluorescence between malignant lesions and normal tissues after oral administration of 5ALA. Malignant lesions on the pleural surface emitted pink autofluorescence in contrast to the green autofluorescence of the surrounding normal tissues. RESULTS: When 28 patients with primary lung cancer were examined according to the degree of pleural infiltration (pl), red fluorescence was confirmed in 10 of 10 patients (100%) with p11-p13 and 5 of 18 patients (27.7%) with p10. The latter 5 patients had been diagnosed with PL1 preoperatively or intraoperatively. CONCLUSION: This system achieved accurate localization of malignant lesions, suggesting that it may also be applicable to photodynamic therapy.

Predicting the "usefulness" of 5-ALA-derived tumor fluorescence for fluorescence-guided resections in pediatric brain tumors: a European survey.

BACKGROUND: Five-aminolevulinic acid (Gliolan, medac, Wedel, Germany, 5-ALA) is approved for fluorescence-guided resections of adult malignant gliomas. Case reports indicate that 5-ALA can be used for children, yet no prospective study has been conducted as of yet. As a basis for a study, we conducted a survey among certified European Gliolan users to collect data on their experiences with children. METHODS: Information on patient characteristics, MRI characteristics of tumors, histology, fluorescence qualities, and outcomes were requested. Surgeons were further asked to indicate whether fluorescence was "useful", i.e., leading to changes in surgical strategy or identification of residual tumor. Recursive partitioning analysis (RPA) was used for defining cohorts with high or low likelihoods for useful fluorescence. RESULTS: Data on 78 patients <18 years of age were submitted by 20 centers. Fluorescence was found useful in 12 of 14 glioblastomas (85 %), four of five anaplastic astrocytomas (60 %), and eight of ten ependymomas grades II and III (80 %). Fluorescence was found inconsistently useful in PNETs (three of seven; 43 %), gangliogliomas (two of five; 40 %), medulloblastomas (two of eight, 25 %) and pilocytic astrocytomas (two of 13; 15 %). RPA of pre-operative factors showed tumors with supratentorial location, strong contrast enhancement and first operation to have a likelihood of useful fluorescence of 64.3 %, as opposed to infratentorial tumors with first surgery (23.1 %). CONCLUSIONS: Our survey demonstrates 5-ALA as being used in pediatric brain tumors. 5-ALA may be especially useful for contrast-enhancing supratentorial tumors. These data indicate controlled studies to be necessary and also provide a basis for planning such a study.

Cloning of two 5-aminolevulinic acid synthase isozymes HemA and HemO from Rhodopseudomonas palustris with favorable characteristics for 5-aminolevulinic acid production.

5-Aminolevulinic acid (ALA) synthase (ALAS) HemA from non-sulfur photosynthetic bacteria has been used for the ALA bioproduction, whereas the isoenzyme HemT/HemO is less studied and not used for ALA production. Two ALAS-encoding genes, hemA and hemO from Rhodopseudomonas palustris were cloned, purified and characterized. The ALASs had very high specific activity, 3.6 and 2.7 U/mg, respectively, and strong affinity for one of its substrates, succinyl-CoA, K m with values of 11 and 4.4 µM, respectively. HemO retained up to 60 % maximum activity within a broad range of concentrations of hemin, while HemA kept only 20 % at 10 µM hemin. Escherichia coli overexpressing HemA or HemO produced 5.7 and 6.3 g ALA/l, respectively, in a 5 l bioreactor.

Fast-drying multi-laminate bioadhesive films for transdermal and topical drug delivery.

No bioadhesive patch-based system is currently marketed. This is despite an extensive number of literature reports on such systems detailing their advantages over conventional pressure sensitive adhesive-based patches in wet environments and describing successful delivery of a diverse array of drug substances. This lack of proprietary bioadhesive patches is largely due to the fact that such systems are exclusively water-based, meaning drying is difficult. In this paper we describe, for the first time, a novel multiple lamination method for production of bioadhesive patches. In contrast to patches produced using a conventional casting approach, which took 48 hours to dry, bioadhesive films prepared using the novel multiple lamination method were dried in 15 min and were folded into formed patches in a further 10 min. Patches prepared by both methods had comparable physicochemical properties. The multiple lamination method allowed supersaturation of 5-aminolevulinic acid to be achieved in formed patch matrices. However, drug release studies were unable to show an advantage for supersaturation with this particular drug, due to its water high solubility. The multiple lamination method allowed greater than 90% of incorporated nicotine to remain within formed patches, in contrast to the 48% achieved for patches prepared using a conventional casting approach. The procedure described here could readily be adapted for automation by industry. Due to the reduced time, energy and ensuing finance now required, this could lead to bioadhesive patch-based drug delivery systems becoming commercially viable. This would, in turn, mean that pathological conditions occurring in wet or moist areas of the body could now be routinely treated by prolonged site-specific drug delivery, as mediated by a commercially produced bioadhesive patch.

Travelling wave ion mobility mass spectrometry of 5-aminolaevulinic acid, porphobilinogen and porphyrins.

RATIONALE: Human porphyrias, diseases caused by enzyme defects in haem biosynthesis, are characterised by the excessive production, accumulation and excretion of porphyrins and/or 5-aminolaevulinic acid (ALA) and porphobilinogen (PBG). A method for the simultaneous separation, detection and identification of ALA, PBG and porphyrins would greatly facilitate the screening and diagnosis of porphyrias. Such a method would also be invaluable for the biochemical study of the haem, chlorophyll and corrin pathways. METHODS: An aqueous mixture containing ALA, PBG and type I isomer porphyrins was diluted with acetonitrile and infused (10 µL/min) into a Waters Synapt G2 high-definition mass spectrometer, equipped with a Z-Spray electrospray ionisation (ESI) source. Mass spectra were acquired in positive ionisation mode and the optimised ion mobility spectrometry (IMS) conditions were as follows: IMS wave height (V), 40; IMS wave velocity (m/s), 648; IMS gas flow (mL/min) 90.40; helium gas flow (mL/min), 182.60. RESULTS: The IMS drift-time increased with increasing ion mass in the order of ALA, PBG, mesoporphyrin, coproporphyrin I, penta-, hexa- and heptacarboxylic acid porphyrin I and uroporphyrin I. The ESI-IMS-MS spectra shows that PBG could form two different positively charged ions by protonation [M+H](+) , m/z 227, or deprotonation [M - H](+) , m/z 225. The protonated PBG (m/z 227) easily eliminated ammonia in source and the fragment ion (m/z 210) was monitored instead. Doubly charged ions of porphyrins having different drift times from the protonated singly charged molecules were observed in high abundance, providing further structural characterisation. CONCLUSIONS: We have shown, for the first time, an analytical method capable of simultaneously separating haem biosynthetic intermediates and metabolites, for a potential rapid clinical screening method for the porphyrias. IMS-MS allowed the separation of doubly charged porphyrin ions, which will be advantageous for the analysis of natural and synthetic tetrapyrrole compounds, while reducing the misinterpretation of contaminants.

5-Aminolevulinic acid tumor paint and photodynamic therapy for myxofibrosarcoma: an in vitro study.

BACKGROUND: 5-Aminolevulinic acid (5-ALA), a fluorescent contrast agent, has been used for tumor paint and photodynamic therapy (PDT) for various tumors, but its use with soft tissue sarcomas is not well documented. Myxofibrosarcoma, a subtype of soft tissue sarcoma with a high local recurrence rate, may benefit from similar types of treatment. The purpose of this study was to analyze the effects of 5-ALA tumor paint and PDT on a myxofibrosarcoma cell line. METHODS: Tumor paint was assessed by exposing micromass pellets of human adipose-derived stromal (ADS) cells or myxofibrosarcoma (MUG-Myx1) cells to 5-ALA. Cell pellets were then visualized using a microscope at established excitation and emission wavelengths. Corrected total cell fluorescence was calculated per accepted protocols. Photodynamic therapy was similarly assessed by exposing ADS and MUG-Myx1 cells to 5-ALA, with subsequent analysis via flow cytometry and real-time confocal microscopy. RESULTS: The use of 5-ALA tumor paint led to a selective fluorescence in MUG-Myx1 cells. Findings were confirmed by flow cytometry. Interestingly, flow cytometry results showed progressive selective cell death with increasing 5-ALA exposure as a result of the PDT effect. PDT was further confirmed using confocal microscopy, which revealed progressive cellular bubble formation consistent with advancing stages of cell death-a finding that was not seen in control ADS cells. CONCLUSIONS: 5-ALA tumor paint and PDT were successfully used on a human myxofibrosarcoma cell line (MUG-Myx1). Results from this study showed both selective fluorescent tagging and selective cytotoxicity of 5-ALA toward malignant myxofibrosarcoma cells, while sparing benign adipose control cells. This finding was further confirmed in a dramatic time-lapse video, visually confirming active, targeted cell death. 5-ALA's two-pronged application of selective tumor identification and cytotoxicity may transform surgical and medical approaches for treating soft tissue sarcomas.

delta-Aminolevulinic acid-synthesizing enzymes need an RNA moiety for activity.

When Chlamydomonas enzymes that convert glutamate to delta-aminolevulinic acid are separated into three fractions, one of the fractions contains RNA, and the RNA moiety is needed for the enzyme activity.

Association of F18-fluoro-ethyl-tyrosin uptake and 5-aminolevulinic acid-induced fluorescence in gliomas.

PURPOSE: Malignant gliomas are highly infiltrative tumours with a fatal prognosis. F18-fluoroethyl-tyrosine (FET)-positron emission tomography (PET) often reveals a broader extension of these tumours compared with contrast-enhanced magnetic resonance imaging (MRI). Complete resection of the contrast-enhancing lesion is aspired. Fluorescence-guided resection using 5-aminolevulinic acid (5-ALA) improved the extent of resection. In this study, we investigated whether the FET uptake correlates with the extent of resection using 5-ALA-induced fluorescence. METHODS: Thirteen patients who underwent preoperative and postoperative MRI, FET-PET and fluorescence-guided neuronavigated resection were included in this study. The areas in which intraoperative fluorescence terminated the resection were marked. After fusion of PET and MRI, the standardized uptake value (SUV) of FET related to normal brain (SUV(R)) was measured in regions of interest corresponding to resected and remaining tissue, respectively. Receiver-operating characteristic (ROC) curve analysis determined the optimal threshold of the relative SUV anticipating 5-ALA-induced fluorescence. RESULTS: During resection a vivid fluorescence was present in all patients. Histology revealed glioblastomas in 11 cases, an anaplastic astrocytoma in one case and a low-grade astrocytoma in one case. The median FET SUV(R) was higher in areas corresponding to the fluorescent tumour compared with the non-fluorescent normal brain (2.321 vs 1.142, p < 0.0001, t-test). A SUV(R) greater than 1.374 predicted the fluorescence with a sensitivity of 0.87 [95% confidence interval (CI): 0.74-0.94] and a specificity of 0.94 (CI: 0.84-0.99). The area under the ROC curve was 0.9656 (CI: 0.9364-0.9948). CONCLUSIONS: FET uptake predicts the 5-ALA-induced fluorescence in glioma patients. Thus, FET-PET provides useful information for planning glioma resection.

Quantitative determination of 5-aminolaevulinic acid and its esters in cell lysates by HPLC-fluorescence.

The development of a reliable sensitive method for the HPLC determination of 5-aminolaevulinic acid (ALA) and ALA esters in cell lysates is described. The method relies on the quantification of a fluorescent derivative of ALA following its derivatisation with acetylacetone and formaldehyde. Following this procedure it is possible to quantify ALA in cell lysates with no need for pre-purification of the sample. The method has been validated in two ranges of concentration (0.6-65 microM, 0.1-10 microg/mL, and 30-600 microM, 5-100 microg/mL), and has also been extended and validated for the determination of ALA released from ALA prodrugs after acidic hydrolysis.

Characterisation of liposomes containing aminolevulinic acid and derived esters.

Liposomes of different compositions have been designed to improve delivery of aminolevulinic acid (ALA) and its esterified derivatives ALA-Hexyl ester (He-ALA) and ALA-Undecanoyl ester (Und-ALA) for its use in photodynamic therapy (PDT). Egg yolk phosphatidyl choline (PC), phosphatidic acid (PA) and phosphatidyl glycerol (PG) were employed in the preparation of the liposomes. Sonicated vesicles composed of PC, PC-PG (80:20) or PC-PA (80:20) containing ALA or derivatives were obtained and purified by a minicolumn centrifugation method. PC liposomes presented encapsulation percentages around 6% for 2 mM ALA, 13% for 2 mM He-ALA and 51% for 2 mM Und-ALA. The addition of PG or PA to the formulation, resulted in an increased entrapment: 19% for 2 mM ALA, 69% for 2 mM He-ALA and 87% for 2 mM Und-ALA in PC-PG liposomes and 21% for 2 mM ALA, 60% for 2 mM He-ALA and 87% for 2 mM Und-ALA in PC-PA liposomes. Higher concentrations of ALA or derivatives resulted in lower percentages of entrapment. The three formulations containing ALA or derivatives were stable up to 1 week upon storage at 4 degrees C. However, upon dilution with medium, ALA leaked from the liposomes, while on the contrary, He-ALA was highly retained, being therefore a good choice for its use in PDT. The stability of Und-ALA upon dilution could not be tested, but Und-ALA proved to have the highest entrapment efficacy.

Determination of optical properties of human brain tumor tissues from 350 to 1000 nm to investigate the cause of false negatives in fluorescence-guided resection with 5-aminolevulinic acid.

The optical properties of human brain tumor tissues, including glioblastoma, meningioma, oligodendroglioma, and metastasis, that were classified into "strong," "vague," and "unobservable" fluorescence by a neurosurgeon were measured and compared. The optical properties of the tissues were measured with a double integrating sphere and the inverse Monte Carlo technique from 350 to 1000 nm. Using reasons of ex-vivo measurement, the optical properties at around 420 nm were potentially affected by the hemoglobin content in tissues. Significant differences were not observed between the optical properties of the glioblastoma regions with "strong" and "unobservable" fluorescence. Sections of human brain tumor tissue with "strong" and "unobservable" fluorescence were stained with hematoxylin and eosin. The cell densities [mean ± standard deviation (S.D.)] in regions with "strong" and "unobservable" fluorescence were 31 ± 9 × 102 per mm2 and 12 ± 4 × 102 per mm2, respectively, which is a statistically significant difference. The higher fluorescence intensity is associated with higher cell density. The difference in cell density modified the scattering coefficient yet it does not lead to significant differences in the reduced scattering coefficient and thus does not affect the propagation of the diffuse fluorescent light. Hence, the false negatives, which mean a brain tumor only shows "unobservable" fluorescence and is hence classified incorrectly as nontumor, in using 5-ALA for detection of human glioblastoma do not result from the differences in optical properties of human brain glioblastoma tissues. Our results suggest that the primary cause of false negatives may be a lack of PpIX or a low accumulation of PpIX.

Liquid chromatography assay for 5-aminolevulinic acid: application to in vitro assessment of skin penetration via Dermaportation.

The purpose of the present study was to develop a reverse-phase high performance liquid chromatographic (HPLC) assay for quantifying 5-aminolevulinic acid (ALA). The assay was applied to study the skin permeation of ALA and the influence of a novel skin penetration enhancement technology. Separation was achieved utilizing a Phenomenex Jupiter C(18) column following fluorescence derivatization with fluorescamine. The assay was linear (r(2)>0.99) with a minimum limit of quantitation of 400 ng/mL. The inter- and intraday variation was 1.6 and 0.9% at the lower end of the linear range and 1.5 and 1.9% at the upper end, respectively. The HPLC assay and fluorescence derivatization procedure is sensitive, simple, rapid, accurate and reproducible and offers advantages with regard to stability of ALA in comparison to other fluorescence derivatization methods. Results from the preliminary skin permeation study demonstrated substantial skin penetration of ALA only when applied with Dermaportation as a skin penetration enhancement device.

Pharmaceutical analysis of 5-aminolevulinic acid in solution and in tissues.

Quantification of 5-aminolevulinic acid (ALA) in solution, and methods used to achieve this, have been extensively reported in the literature. However, validated methods have only rarely been presented and never have methods been compared. Due to a necessity in drug delivery research for optimised and validated methods for determination of ALA in solution, this paper compares, for the first time, two such methods validated to International Conference on Harmonisation (ICH) standards. Of major importance, derivatisation of ALA with acetyl acetone and formaldehyde was found to be more suitable for routine fluorimetric HPLC analysis of ALA than derivatisation with o-phthaldialdehyde and 2-mercaptoethanol. This former method was successfully utilised in the comparison of in vitro drug release from a proprietary ALA cream and a novel bioadhesive patch-based system. In addition, determination of ALA in tissue is necessary to compare different topical formulations, in terms of their ability to deliver the drug successfully, and different tissue types, to assess their barrier properties to penetration of the drug. Consequently, this paper also describes the use of liquid scintillation spectroscopy as an analytical tool for rapid, convenient and routine quantification of ALA in tissue and determination of penetration depth following topical application of creams and patches.

A gas chromatography-mass spectrometry method for the determination of delta-aminolevulinic acid in plant leaves.

Delta-aminolevulinic (δ-ALA) acid is an important intermediate for tetrapyrroles biosynthesis and it has recently received great attention in plant physiology and human toxicology. However, the colorimetric method which is the most common method for determination of δ-ALA is time consuming and is not specific. In this study, a method for determination of δ-ALA in plant tissues was developed based on the trimethylsilyl (TMS) derivative of the pyrrole formed from the reaction of δ-ALA with ethyl acetoacetate via Knorr condensation. The δ-ALA in the HCl extract was reacted with ethyl acetoacetate to form a pyrrole. Then, the pyrrole compound was extracted using ethyl acetate and the solvent was evaporated to dryness. The dried sample was derivatized to its TMS ester and analyzed using GC-MS. The concentration of δ-ALA in citrus leaves incubated with levulinic acid was also determined by the conventional colorimetric method. The linear range was 10-200ppm in the full scan mode and 0.1-20ppm in the selected ion monitoring (SIM). The limit of detection was 6ppm in the full scan and 0.05ppm in SIM mode, representing a four-fold increase in sensitivity compared to the colorimetric method. The GC-MS method developed in this study is simple, accurate, sensitive, and could also be used to measure δ-ALA in other biological samples.

Stability of 5-aminolevulinic acid on dried urine filter paper for a diagnostic marker of tyrosinemia type I.

The chemical diagnosis of tyrosinemia type I generally involves the detection of succinylacetone (SA) in patient urine. However, 5-aminolevulinate (5ALA), which accumulates due to succinylacetone's inhibition of porphyrin synthesis, can also be used as diagnostic metabolites. Here we examined the stabilities of these markers on dried urine filter paper. After two weeks at room temperature, the succinylacetone was 10% of its original level, but over 80% of 5-aminolevulinate remained. Thus, although insufficient succinylacetone was recovered from dried urine filter paper to diagnose tyrosinemia type I, 5-aminolevulinate was readily detected, permitting the diagnosis.

Analysis of pyrazine 2,5-dipropionic acid in 5-aminolevulinic acid-loaded urological and topical delivery vehicles: methodology and assay validation.

Photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA) is a novel treatment method with much potential benefit for cancer detection and eradication. Formulation into drug delivery systems, such as aqueous solutions and emulsion based creams is complicated by its rapid dimerisation to pyrazine 2,5-dipropionic acid (PY); a compound with scant documentation in terms of toxicity and effect during PDT. This degradation is especially noticeable, where pH is adjusted upwards to avoid local irritation. A good case in point is bladder instillation of ALA for treatment and diagnosis of urothelial neoplasia. This work describes a rapid and validated HPLC method designed to assess the formation of PY in ALA-loaded vehicles. PY eluted as a single peak (Rt=5.0 min) with good intra- and inter-day reproducibility and limits of detection and quantification found to be 0.01 and 0.04 microg ml(-1), respectively. Sample stability for upto 16 h was demonstrated, allowing autoinjection cycles to be performed. PY formation was detected in typical buffers used for bladder instillation after 6 h of storage, emphasising the need to use these preparations immediately upon manufacture if intended for photodynamic purposes. Moreover, upto 2.35% (w/w) PY was detected in artificial urine after 6 h storage at ambient temperature indicating that formation in vivo is likely to occur once bladder instillations are in situ and exposed to endogenous urine. As a result, ALA instillation times should be kept to the minimum needed for safe and successful treatment or diagnosis. PY extraction from semi-solid devices approached 100% efficiency demonstrating that the reported assay is suitable for evaluating stability of novel dosage forms intended for ALA delivery.

[Study of a multivariate statistical 5-ALA-based discrimination method for fluorescence spectra of colonic tissue of SD rats].

In order to diagnosis colon early cancer with laser-induced 5-ALA-PpIX fluorescence spectra, a multivariate statistical method to distinguish these fluorescence spectra acquired in vivo was developed. 343 spectra were collected from 8 normal SD rats, and 20 1,2-DMH-induced SD colon cancer models, and 12 second generation rats of induced rats. 150 min after trail intravenous injections of 5-ALA at a dose of 25 mg x kg(-1) BW, fluorescence spectra excited with 370 nm Ti-laser were collected in vivo. All spectra were divided into a calibration group and a prediction group. After preprocessing, 4 principal components were extracted with PCA. And then, discrimination models were built by stepwise multivariate logistic regression (SMLR) on calibration group. 3 pathological styles were combined each other, and then 3 SMLR models were derived. Normal tissues were classified from early cancers and advanced cancers with sensitivity of 100% and 98.4%, and specificity of 96% and 100%, and accuracy of 98% and 99.2% on prediction group, respectively. The multivariate statistical discrimination method of PCA and SMLR together can effectively distinguish normal tissues from early cancers and advanced cancers with high sensitivity and specificity by means of systemic 5-ALA at low dose. Laser induced fluorescence 5-ALA-based technique is promising for the detection of colonic early cancer.