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 In a trial involving 12 adult subjects with mild to moderate actinic keratosis (AK) and at least 10 AK lesions on the face or forehead, the pharmacokinetics (PK) of aminolevulinic acid (ALA) and protoporphyrin IX (PpIX) were evaluated. Subjects received a single application of one tube of ALA (2 g) under closed-circuit conditions for 3 hours, followed by photodynamic therapy (PDT) covering a total area of 20 cm². The mean ± standard deviation of baseline plasma ALA and PpIX concentrations were 20.16 ± 16.53 ng/mL and 3.27 ± 2.40 ng/mL, respectively. In most subjects, plasma ALA concentrations increased by up to 2.5-fold within the first 3 hours after ALA application. The mean ± standard deviation area under the concentration-time curve (AUC0-t) and maximum concentration (Cmax) of ALA (n=12) after baseline correction were 142.83 ± 75.50 ng·h/mL and 27.19 ± 20.02 ng/mL, respectively. The median time to reach Cmax (Tmax) was 3 hours. ### Topical Solution Two human pharmacokinetic (PK) studies were conducted in subjects with mild to moderate actinic keratosis of the upper extremities, with at least 6 lesions on one upper extremity and at least 12 lesions on the other. The single-dose regimen consisted of two topical applications of ALA solution (each containing 354 mg ALA HCl) directly to the lesion site, followed by a 3-hour occlusion before phototherapy. The first PK study included 29 subjects and assessed the PK parameters of ALA. The baseline-corrected mean ± standard deviation of the maximum concentration (Cmax) of ALA was 249.9 ± 694.5 ng/mL, and the median time to peak concentration (Tmax) was 2 hours after administration. The mean exposure to ALA (expressed as area under the concentration-time curve (AUCt)) was 669.9 ± 1610 ng·hr/mL. The mean elimination half-life (t1/2) of ALA was 5.7 ± 3.9 hours. A second pharmacokinetic (PK) study was conducted in 14 subjects, and PK parameters for ALA and PpIX were determined. In 50% (7/14) of the subjects, the baseline-corrected PpIX concentration was negative in at least 50% of the samples, so the AUC could not be reliably estimated. The baseline-corrected mean ± standard deviation of Cmax for ALA and PpIX were 95.6 ± 120.6 ng/mL and 0.95 ± 0.71 ng/mL, respectively. The median time to peak concentration (Tmax) for ALA and PpIX was 2 hours and 12 hours after administration, respectively. The mean AUCt for ALA was 261.1 ± 229.3 ng·hr/mL. The mean half-life (t1/2) for ALA was 8.5 ± 6.7 hours. ### Oral Solution In 12 healthy subjects, the absolute bioavailability of ALA after administration of the recommended dose of ALA solution was 100.0% ± 1.1, ranging from 78.5% to 131.2%. The median time to peak plasma concentration of ALA was 0.8 hours (range 0.5–1.0 hours).
Route of Excretion
In 12 healthy subjects, the rate of maternal ALA excretion in urine within 12 hours following administration of the recommended dose of aminolevulinic acid (ALA) solution was 34 ± 8% (mean ± standard deviation), ranging from 27% to 57%.
Volume of Distribution
In healthy volunteers, the volume of distribution of aminolevulinic acid was 9.3 ± 2.8 L for intravenous administration and 14.5 ± 2.5 L for oral administration. [11961050]

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Metabolism/Metabolites
Exogenous aminolevulinic acid (ALA) is metabolized to protoporphyrin IX (PpIX), but the proportion of ALA metabolized to PpIX is unknown. The mean plasma AUC of PpIX is less than 6% of that of ALA. Following topical administration, the drug is synthesized in situ into protoporphyrin IX within the skin. Half-life: The mean half-life after oral administration was 0.70 ± 0.18 hours, and the mean half-life after intravenous administration was 0.83 ± 0.05 hours. Biological half-life: The mean elimination half-life (t1/2) of the topical aminolevulinic acid solution was 5.7 ± 3.9 hours, and the mean half-life of the oral solution was 0.9 ± 1.2 hours. In another pharmacokinetic study involving 6 healthy volunteers, using a 128 mg dose, the mean half-life after oral administration was 0.70 ± 0.18 hours, and the mean half-life after intravenous administration was 0.83 ± 0.05 hours.

Toxicity Overview
Based on the hypothesized mechanism of action, photosensitization following topical application of aminolevulinic acid (ALA) solution is achieved through the metabolism of ALA into protoporphyrin IX (PpIX), which accumulates in the skin where aminolevulinic acid is applied. Upon exposure to light of appropriate wavelength and energy, the accumulated PpIX undergoes a photodynamic reaction, a cytotoxic process dependent on the simultaneous presence of light and oxygen. Light absorption leads to the excited state of the porphyrin molecule, followed by spin shift of PpIX towards molecular oxygen to generate singlet oxygen, which can further react to generate superoxide anions and hydroxyl radicals. The use of aminolevulinic acid for photosensitization of actinic keratosis lesions, combined with irradiation using the BLU-UTM blue light photodynamic therapy device (BLU-U), forms the basis of aminolevulinic acid photodynamic therapy (PDT).

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Effects during Pregnancy and Lactation
◉ Overview of Use During Lactation
Currently, there is no information regarding oral administration of aminolevulinic acid during lactation. To minimize infant exposure, breastfeeding should be suspended for 24 hours after oral administration. Due to extremely low systemic absorption, breastfeeding is not expected to result in local exposure of the infant to aminolevulinic acid. Aminolevulinic acid-induced photodynamic therapy has been successfully used to treat various nipple skin lesions. This therapy appears to help maintain nipple anatomy, thus facilitating breastfeeding.
◉ Effects on breastfed infants
No relevant published information found as of the revision date.
◉ Effects on breastfeeding and breast milk
No relevant published information found as of the revision date.

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Route of administration
Oral bioavailability is 50-60%.

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Protein binding
In in vitro studies, after using aminolevulinic acid (ALA) at concentrations reaching approximately 25% of the maximum plasma concentration of ALA solution at the recommended dose, the average protein binding rate 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 salt is the monohydrochloride salt of 5-aminolevulinic acid. It is metabolized to protoporphyrin IX, a photosensitizing compound that accumulates in the skin. It is used in conjunction with blue light irradiation to treat mild to moderate actinic keratosis of the face or scalp. It has a dual role as an antitumor drug, photosensitizer, dermatological drug, and prodrug. It contains 5-aminolevulinic acid.
Aminolevulinic acid salt is the hydrochloride form of aminolevulinic acid (an aminoketone) used for topical photosensitization therapy. Aminolevulinic acid (ALA) is a metabolic prodrug that can be converted into the photosensitizer protoporphyrin IX (PpIX), which accumulates intracellularly. When exposed to light of an appropriate wavelength (red or blue), PpIX catalyzes the production of singlet oxygen, an endotoxin that can further react to generate superoxide anions and hydroxyl radicals. This leads to cytotoxic effects.
PpIX is an intermediate compound produced from succinyl-CoA and glycine during heme synthesis. It is used as a photochemotherapy for actinic keratosis.

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Drug Indications
Gliolan is indicated for adult patients for visualization of malignant tissue during surgery for malignant gliomas (WHO Grade III and IV).
For the treatment of mild to moderate actinic keratosis (Olsen Grade 1 to 2; see Section 5.1) of the adult face and scalp, as well as cancerous areas. This study aims to explore treatment options with 5-aminolevulinic acid for adult patients with superficial and/or nodular basal cell carcinoma who are unsuitable for surgical treatment due to potential treatment-related complications and/or poor cosmetic outcomes.

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Background: 5-Aminolevulinic acid is a non-fluorescent prodrug that can lead to the accumulation of fluorescent porphyrins within malignant glioma cells—a finding being used for intraoperative identification and resection of these tumors. This study aims to evaluate the impact of fluorescence-guided resection using 5-aminolevulinic acid on tumor radicality, progression-free survival, overall survival, and complications. Methods: 322 patients aged 23 to 73 years with suspected malignant gliomas and suitable for complete enhanced tumor resection were randomly assigned to two groups: one group received fluorescence-guided resection with 20 mg/kg body weight of 5-aminolevulinic acid (n=161), and the other group received conventional white light microsurgery (n=161). The primary endpoint was the number of patients with no enhancement on early MRI (i.e., MRI performed within 72 hours postoperatively) and 6-month progression-free survival as assessed by MRI. Secondary endpoints included residual tumor volume on postoperative MRI, overall survival, neurological deficits, and toxicities. We report the results of an interim analysis that included 270 patients (139 treated with 5-aminolevulinic acid and 131 treated with white light), excluding patients whose histological and radiological results, as assessed by the center reviewer (unaware of the treatment allocation), did not meet the inclusion criteria; the interim analysis results led to the termination of the study according to the protocol. Both the primary and secondary endpoints were analyzed in the full analysis population using an intention-to-treat approach. This study was registered at http://www.clinicaltrials.gov, registration number NCT00241670. Results: The median follow-up time was 35.4 months (95% CI 1.0–56.7). Of the 139 patients treated with 5-aminolevulinic acid, 90 (65%) of enhancing tumors were completely resected; compared to only 47 (36%) of the 131 patients treated with white light therapy (between groups: 29% [95% CI 17–40], p<0.0001). Patients treated with 5-aminolevulinic acid had a longer 6-month progression-free survival than those treated with white light therapy (41.0% [32.8–49.2] vs 21.1% [14.0–28.2]; between groups: 19.9% [9.1–30.7], p=0.0003, Z-test). There were no differences among the groups in the frequency of serious adverse events or adverse events of any organ system category reported within 7 days postoperatively. Conclusion: Tumor fluorescence derived from 5-aminolevulinic acid can more thoroughly remove enhancing tumors, thereby improving progression-free survival in patients with malignant gliomas. [1]

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Background: 5-aminolevulinic acid (5-ALA) is a natural amino acid widely used in cancer treatment due to its tumor-specific metabolic pathway characteristics. This study shows that 5-ALA induces ferroptosis through glutathione peroxidase 4 (GPX4) and heme oxygenase 1 (HMOX1) and has antitumor effects in esophageal squamous cell carcinoma (ESCC). Methods: The expression of GPX4 and HMOX1 in pathological specimens from 97 ESCC patients was detected and prognostic analysis was performed. The role of 5-aminolevulinic acid (5-ALA) in in vitro ferroptosis was evaluated using real-time polymerase chain reaction (RT-PCR), RNA microarray and Western blot analysis. Furthermore, this study also examined 5-ALA-treated cell lines using the ferroptosis inhibitor ferrostatin-1 and a lipid peroxidation reagent. Finally, the role of 5-ALA was further confirmed by its effect in a mouse model of subcutaneous xenograft esophageal squamous cell carcinoma (ESCC). The results showed that GPX4 upregulation and HMOX1 downregulation are factors contributing to poor prognosis in ESCC. In RNA microarray analysis of KYSE30 cells, ferroptosis was one of the most frequently induced pathways; 5-ALA treatment inhibited GPX4 expression and upregulated HMOX1 expression. These results were validated by RT-PCR and Western blotting experiments. In addition, 5-ALA leads to increased lipid peroxidation and exerts anti-tumor effects in various cancer cell lines, an effect that can be inhibited by the ferroptosis inhibitor ferrostatin-1. In vivo experiments showed that 5-ALA inhibits GPX4 expression and upregulates HMOX1 expression in tumor tissue, thereby leading to tumor volume reduction. Conclusion: 5-ALA induces ferroptosis in esophageal squamous cell carcinoma (ESCC) cells by regulating the expression of GPX4 and HMOX1. Therefore, 5-ALA may be a promising novel 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.)