Endogenous Metabolite

It is being researched for intraoperative tumor identification and resection if 5-aminolevulinic acid hydrochloride (ALA), a nonfluorescent prodrug, stimulates the formation of fluorescent porphyrins in malignant glioma cells. 35.4 months was the median follow-up period (95% CI 1.0-56.7). Ninety of the 139 patients (65%) treated with 5-aminolevulinic acid had all of their contrast-enhancing tumors fully removed, while only 36 of the 131 patients (36%) treated with white light had this outcome (the difference between the groups was 29% [95% CI 17-40], p < 0.0001). The 6-month progression-free survival was higher in patients treated with 5-aminolevulinic acid than in patients treated with white light (41.0% [32.8-49.2] vs 21.1% [14.0-28.2]; between-group difference 19.9% [9.1-30.7], p= 0.0003, Z test) [1]. It has been demonstrated that 5-ALA by itself is insufficient to provide complete excision without running the risk of neurologic decline following surgery. Furthermore, iMRI in conjunction with functional neuronavigation is notably superior to the 5-ALA resection approach for functional grade III gliomas [2].

Despite continuing debates around cytoreductive surgery in malignant gliomas, there is broad consensus that increased extent of tumor reduction improves overall survival. However, maximization of the extent of tumor resection is hampered by difficulty in intraoperative discrimination between normal and pathological tissue. In this context, two established methods for tumor visualization, fluorescence guided surgery with 5-ALA and intraoperative MRI (iMRI) with integrated functional neuronavigation were investigated as a dual intraoperative visualization (DIV) approach. Thirty seven patients presumably suffering from malignant gliomas (WHO grade III or IV) according to radiological appearance were included. Twenty-one experimental sequences showing complete resection according to the 5-ALA technique were confirmed by iMRI. Fourteen sequences showing complete resection according to the 5-ALA technique could not be confirmed by iMRI, which detected residual tumor. Further analysis revealed that these sequences could be classified as functional grade II tumors (adjacent to eloquent brain areas). The combination of fluorescence guided resection and intraoperative evaluation by high field MRI significantly increased the extent of tumor resection in this subgroup of malignant gliomas located adjacent to eloquent areas from 61.7% to 100%; 5-ALA alone proved to be insufficient in attaining gross total resection without the danger of incurring postoperative neurological deterioration. Furthermore, in the case of functional grade III gliomas, iMRI in combination with functional neuronavigation was significantly superior to the 5-ALA resection technique. The extent of resection could be increased from 57.1% to 71.2% without incurring postoperative neurological deficits[2].

Expression of GPX4 and HMOX1 in pathologic specimens of 97 ESCC patients was examined, and prognostic analyses were performed. Real-time polymerase chain reaction (RT-PCR), RNA microarray, and Western blotting analyses were used to evaluate the role of 5-ALA in ferroptosis in vitro. Ann Surg Oncol. 2021 Jul;28(7):3996-4006. https://pubmed.ncbi.nlm.nih.gov/33210267/

Dual Intraoperative Visualization (DIV) protocol[2]
Tumor volumetry was performed immediately prior to surgery. Tumor resection was then performed using the 5-ALA signal alone with the absence of a visible signal defining completeness of resection. This determination was carried out by the primary surgeon at all times. Functional neuronavigation data was intermittently projected to prevent inadvertent damage to functional brain areas. At the end of each stage of resection, the tumor cavity was systematically inspected to exclude residual tumor. Once the 5-ALA signal was undetectable, an iMRI scan was performed. If the extent of resection was confirmed, the decision to conclude the surgery was taken by the primary surgeon. Otherwise, the residual tumor volume was re-segmented and resection continued according to the neuronavigation. In all such cases the 5-ALA signal was redetected during further surgery once either the thin intervening layer of “healthy” brain parenchyma was removed and/or the viewing angle subsequently optimized. This procedure was repeated until the 5-ALA signal was no longer detectable, and the corresponding absence of contrast-enhancing tumor corroborated by iMRI. The additionally resected tissue detected by the iMRI was also analyzed by an experienced neuropathologist, confirming pathological glioma cell infiltration. In the event of persistence of 5-ALA in areas shown to be functional by the neuronavigation data, further surgery in the corresponding direction was intentionally terminated.

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In addition, this study used ferrostatin-1, a ferroptosis inhibitor, and a lipid peroxidation reagent against cell lines treated with 5-ALA. Finally, the role of 5-ALA was confirmed by its effect on an ESCC subcutaneous xenograft mouse model. Ann Surg Oncol. 2021 Jul;28(7):3996-4006. https://pubmed.ncbi.nlm.nih.gov/33210267/

Absorption
Oral bioavailability is 50-60%. ### **Topical gel** Pharmacokinetics (PK) of aminolevulinic acid (ALA) and PpIX was evaluated in a trial of 12 adult subjects with mild to moderate AK with at least 10 AK lesions on the face or forehead. A single dose of one entire tube of ALA (2 grams) was applied under occlusion for 3 hours followed by photodynamic therapy (PDT) to a total area of 20 cm2. The mean ± SD baseline plasma ALA and PpIX concentrations were 20.16 ± 16.53 ng/mL and 3.27 ± 2.40 ng/mL, respectively. In most subjects, an up to 2.5-fold increase of ALA plasma concentrations was observed during the first 3 hours after ALA application. The mean ± SD area under the concentration time curve (AUC0-t) and maximum concentration (Cmax) for baseline corrected ALA (n=12) were 142.83 ± 75.50 ng.h/mL and 27.19 ± 20.02 ng/mL, respectively. The median Tmax (time at which Cmax occurred) was 3 hours. ### **Topical solution** Two human pharmacokinetic (PK) studies were conducted in subjects with minimally to moderately thick actinic keratoses on the upper extremities, having at least 6 lesions on one upper extremity and at least 12 lesions on the other upper extremity. A single dose comprising of two topical applications of ALA topical solution (each containing 354 mg ALA HCl) were directly applied to the lesions and occluded for 3 hours prior to light treatment. The first PK study was conducted in 29 subjects and PK parameters of ALA were assessed. The baseline corrected mean ± SD of the maximum concentration (Cmax) of ALA was 249.9 ± 694.5 ng/mL and the median Tmax was 2 hours post dose. The mean ± SD exposure to ALA, as expressed by area under the concentration time curve (AUCt) was 669.9 ± 1610 ng·hr/mL. The mean ± SD elimination half-life (t1/2) of ALA was 5.7 ± 3.9 hours. A second PK study was conducted in 14 subjects and PK parameters of ALA and PpIX were measured. The baseline corrected PpIX concentrations were negative in at least 50% of samples in 50% (7/14) subjects and AUC could not be estimated reliably. The baseline-corrected mean ± SD of Cmax for ALA and PpIX was 95.6 ± 120.6 ng/mL and 0.95 ± 0.71 ng/mL, respectively. The median Tmax of ALA and PpIX was 2 hours post dose and 12 hours post dose, respectively. The mean AUCt of ALA was 261.1 ± 229.3 ng·hr/mL. The mean ± SD t1/2 of ALA was 8.5 ± 6.7 hours. ### **Oral solution** In 12 healthy subjects, the absolute bioavailability of ALA following the recommended dose of ALA solution was 100.0% + 1.1 with a range of 78.5% to 131.2%. Maximum ALA plasma concentrations were reached with a median of 0.8 hour (range 0.5 – 1.0 hour).

Route of Elimination
In 12 healthy subjects, excretion of parent aminolevulinic acid (ALA) in urine in the 12 hours following administration of the recommended dose of ALA solution was 34 + 8% (mean + std dev) with a range of 27% to 57%.

Volume of Distribution
In healthy volunteers, the administration of aminolevulinic acid resulted in a volume of distribution of 9.3 ± 2.8 L intravenously and 14.5 ± 2.5 orally.[11961050]

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Metabolism / Metabolites
Exogenous aminolevulinic acid (ALA) is metabolized to PpIX, but the fraction of administered ALA that is metabolized to PpIX is unknown. The average plasma AUC of PpIX is less than 6% of that of ALA.
Following topical administration, synthesis into protoporphyrin IX takes place in situ in the skin. Half Life: Mean half-life is 0.70 ± 0.18 h after the oral dose and 0.83 ± 0.05 h after the intravenous dose.

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Biological Half-Life
The mean ± SD elimination half-life (t1/2) of aminolevulinic acid was 5.7 ± 3.9 hours for the topical solution formulation and the mean half-life was 0.9 ± 1.2 hours for the oral solution formulation. In another pharmacokinetic studies with 6 healthy volunteers using a 128 mg dose, the mean half-life was 0.70 ± 0.18 h after the oral dose and 0.83 ± 0.05 h after the intravenous dose.

Toxicity Summary
According to the presumed mechanism of action, photosensitization following application of aminolevulinic acid (ALA) topical solution occurs through the metabolic conversion of ALA to protoporphyrin IX (PpIX), which accumulates in the skin to which aminolevulinic acid has been applied. When exposed to light of appropriate wavelength and energy, the accumulated PpIX produces a photodynamic reaction, a cytotoxic process dependent upon the simultaneous presence of light and oxygen. The absorption of light results in an excited state of the porphyrin molecule, and subsequent spin transfer from PpIX to molecular oxygen generates singlet oxygen, which can further react to form superoxide and hydroxyl radicals. Photosensitization of actinic (solar) keratosis lesions using aminolevulinic acid, plus illumination with the BLU-UTM Blue Light Photodynamic Therapy Illuminator (BLU-U), is the basis for aminolevulinic acid photodynamic therapy (PDT).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the use of oral aminolevulinic acid during breastfeeding. To minimize exposure of the infant, breastfeeding can be withheld for 24 hours after an oral dose. Breastfeeding is not expected to result in exposure of the child to topical aminolevulinic acid due to negligible systemic absorption. Aminolevulinic acid-induced photodynamic therapy has been used successfully to treat various skin lesions of the nipple. This treatment appeared to preserve nipple anatomy for breastfeeding.

◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.

◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Exposure Routes
Oral bioavailability is 50-60%.

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Protein Binding
In in vitro experiments using aminolevulinic acid (ALA) concentrations up to approximately 25% of the maximal concentration that occurs in plasma following the recommended dose of ALA solution, the mean protein binding of ALA was 12%.

[1]. Stummer, W., et al., Fluorescence-guided surgery with 5-aminolevulinic acid for resection of malignant glioma: a randomised controlled multicentre phase III trial. Lancet Oncol, 2006. 7(5): p. 392-401.
[2]. Eyupoglu, I.Y., et al., Improving the extent of malignant glioma resection by dual intraoperative visualization approach. PLoS One, 2012. 7(9): p. e44885.

5-aminolevulinic acid hydrochloride is a hydrochloride that is the monohydrochloride of 5-aminolevulinic acid. It is metabolised to protoporphyrin IX, a photoactive compound which accumulates in the skin. Used in combination with blue light illumination for the treatment of minimally to moderately thick actinic keratosis of the face or scalp. It has a role as an antineoplastic agent, a photosensitizing agent, a dermatologic drug and a prodrug. It contains a 5-ammoniolevulinic acid.

Aminolevulinic Acid Hydrochloride is the hydrochloride salt form of aminolevulinic acid, an aminoketone, used for local photosensitizing therapy. Aminolevulinic acid (ALA) is a metabolic pro-drug that is converted into the photosensitizer protoporphyrin IX (PpIX), which accumulates intracellularly. Upon exposure to light of appropriate wavelength (red, or blue), PpIX catalyzes oxygen to singlet oxygen, an intracellular toxin, which can further react to form superoxide and hydroxyl radicals. This leads to cellular cytotoxic effects.
A compound produced from succinyl-CoA and GLYCINE as an intermediate in heme synthesis. It is used as a PHOTOCHEMOTHERAPY for actinic KERATOSIS.

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Drug Indication
Gliolan is indicated in adult patients for visualisation of malignant tissue during surgery for malignant glioma (World Health Organization grade III and IV).

Treatment of actinic keratosis of mild to moderate severity on the face and scalp (Olsen grade 1 to 2; see section 5. 1) and of field cancer ization in adults. Treatment of superficial and/or nodular basal cell carcinoma unsuitable for surgical treatment due to possible treatment-related morbidity and/or poor cosmetic outcome in adults.

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Background: 5-Aminolevulinic acid is a non-fluorescent prodrug that leads to intracellular accumulation of fluorescent porphyrins in malignant gliomas-a finding that is under investigation for intraoperative identification and resection of these tumours. We aimed to assess the effect of fluorescence-guided resection with 5-aminolevulinic acid on surgical radicality, progression-free survival, overall survival, and morbidity. Methods: 322 patients aged 23-73 years with suspected malignant glioma amenable to complete resection of contrast-enhancing tumour were randomly assigned to 20 mg/kg bodyweight 5-aminolevulinic acid for fluorescence-guided resection (n=161) or to conventional microsurgery with white light (n=161). The primary endpoints were the number of patients without contrast-enhancing tumour on early MRI (ie, that obtained within 72 h after surgery) and 6-month progression-free survival as assessed by MRI. Secondary endpoints were volume of residual tumour on postoperative MRI, overall survival, neurological deficit, and toxic effects. We report the results of an interim analysis with 270 patients in the full-analysis population (139 assigned 5-aminolevulinic acid, 131 assigned white light), which excluded patients with ineligible histological and radiological findings as assessed by central reviewers who were masked as to treatment allocation; the interim analysis resulted in termination of the study as defined by the protocol. Primary and secondary endpoints were analysed by intention to treat in the full-analysis population. The study is registered at http://www.clinicaltrials.gov as NCT00241670. Findings: Median follow-up was 35.4 months (95% CI 1.0-56.7). Contrast-enhancing tumour was resected completely in 90 (65%) of 139 patients assigned 5-aminolevulinic acid compared with 47 (36%) of 131 assigned white light (difference between groups 29% [95% CI 17-40], p<0.0001). Patients allocated 5-aminolevulinic acid had higher 6-month progression free survival than did those allocated white light (41.0% [32.8-49.2] vs 21.1% [14.0-28.2]; difference between groups 19.9% [9.1-30.7], p=0.0003, Z test). Groups did not differ in the frequency of severe adverse events or adverse events in any organ system class reported within 7 days after surgery. Interpretation: Tumour fluorescence derived from 5-aminolevulinic acid enables more complete resections of contrast-enhancing tumour, leading to improved progression-free survival in patients with malignant glioma.[1]

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Background: Due to its tumor-specific metabolic pathway characteristics, 5-aminolevulinic acid (5-ALA) is a natural amino acid widely used in cancer treatment. The current study, demonstrated that 5-ALA induced ferroptosis via glutathione peroxidase 4 (GPX4) and heme oxygenase 1 (HMOX1) and had an antitumor effect in esophageal squamous cell carcinoma (ESCC). Methods: Expression of GPX4 and HMOX1 in pathologic specimens of 97 ESCC patients was examined, and prognostic analyses were performed. Real-time polymerase chain reaction (RT-PCR), RNA microarray, and Western blotting analyses were used to evaluate the role of 5-ALA in ferroptosis in vitro. In addition, this study used ferrostatin-1, a ferroptosis inhibitor, and a lipid peroxidation reagent against cell lines treated with 5-ALA. Finally, the role of 5-ALA was confirmed by its effect on an ESCC subcutaneous xenograft mouse model. Results: The study showed that upregulation of GPX4 and downregulation of HMOX1 were poor prognostic factors in ESCC. In an RNA microarray analysis of KYSE30, ferroptosis was one of the most frequently induced pathways, with GPX4 suppressed and HMOX1 overexpressed by 5-ALA treatment. These findings were verified by RT-PCR and Western blotting. Furthermore, 5-ALA led to an increase in lipid peroxidation and exerted an antitumor effect in various cancer cell lines, which was inhibited by ferrostatin-1. In vivo, 5-ALA suppressed GPX4 and overexpressed HMOX1 in tumor tissues and led to a reduction in tumor size. Conclusions: Modulation of GPX4 and HMOX1 by 5-ALA induced ferroptosis in ESCC. Thus, 5-ALA could be a promising new therapeutic agent for ESCC. Reference: Ann Surg Oncol. 2021 Jul;28(7):3996-4006.

C5H9NO3.H3O4P

229.12508

229.035

C, 26.21; H, 5.28; N, 6.11; O, 48.88; P, 13.52

868074-65-1

5451-09-2 (HCl);106-60-5 (free);868074-65-1 (phosphate);

24737828

Typically exists as solid at room temperature

5

8

4

14

171

0

O=C(CCC(CN)=O)O.O=P(O)(O)O

XWNWBYZHOAIHTK-UHFFFAOYSA-N

InChI=1S/C5H9NO3.H3O4P/c6-3-4(7)1-2-5(8)9;1-5(2,3)4/h1-3,6H2,(H,8,9);(H3,1,2,3,4)

5-amino-4-oxopentanoic acid;phosphoric acid

Aminolevulinic acid phosphate; 868074-65-1; 5-Aminolevulinic acid phosphate; Pentanoic acid, 5-amino-4-oxo-, phosphate (1:1); UNII-FM8DCR39GH; delta-Aminolevulinic acid phosphate; FM8DCR39GH; Aminolevulinic acid phosphate [INCI];

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)