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| Targets |
The molecular target of Asfotase alfa is the alkaline phosphatase (ALP) deficiency that underlies hypophosphatasia. Hypophosphatasia (HPP) is caused by mutations in the ALPL gene, leading to deficient activity of tissue-nonspecific alkaline phosphatase (TNAP). Asfotase alfa replaces the missing TNAP enzyme, which is essential for bone mineralization. The deca-aspartate domain targets the fusion protein to the bone microenvironment. The mechanism involves the hydrolysis of inorganic pyrophosphate (PPi), an inhibitor of mineralization, thereby promoting physiological bone mineralization.
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| ln Vitro |
In vitro, Asfotase alfa (ENB-0040) functions as a bone-targeted alkaline phosphatase. It demonstrates enzymatic activity similar to the native TNAP enzyme, catalyzing the conversion of orthophosphoric monoester and water to an alcohol and orthophosphate. The deca-aspartate (D10) domain enables binding to hydroxyapatite in bone, with higher affinity for calcified tissues. It has been shown to hydrolyze inorganic pyrophosphate (PPi) and pyridoxal 5'-phosphate (PLP), two natural substrates of TNAP, thereby improving the mineralization process in vitro.
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| ln Vivo |
The dose-response relationships of asfotase alfa (0.5-8.2 mg/kg; sc once daily for 43 days) provide significant evidence for the pharmacological efficacy for HPP[1].
In vivo, Asfotase alfa increases the survival rate, bone mineralization, and bone length, and prevents mineralization defects of the feet, rib cage, lower limbs, and jaw bones in Akp2-/- knockout mice, a model of severe infantile hypophosphatasia. In Akp2-/- mice, treatment with Asfotase alfa at doses of 0.5, 2.0, or 8.2 mg/kg (s.c., once daily for 43 days) shows a dose-response relationship for survival, body weight, bone length, and mineralization, demonstrating robust efficacy. |
| Enzyme Assay |
A standard biochemical assay for Asfotase alfa is an alkaline phosphatase (ALP) enzyme activity assay. The assay uses a chromogenic substrate such as para-nitrophenyl phosphate (pNPP). The reaction mixture contains Asfotase alfa (or tissue sample), pNPP, and an appropriate buffer (e.g., 1 M diethanolamine, pH 9.8, with 0.5 mM MgCl2). The reaction is incubated at 37degC for 15-30 minutes. The production of para-nitrophenol (pNP) is measured by absorbance at 405 nm. One unit of ALP activity is defined as the amount of enzyme that hydrolyzes 1 micromol of pNPP per minute. This assay is used to confirm the enzymatic activity of the drug.
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| Cell Assay |
An in vitro cellular assay for Asfotase alfa is a mineralization assay using primary osteoblasts or mesenchymal stem cells (MSCs). Cells are cultured in osteogenic differentiation media (containing beta-glycerophosphate and ascorbic acid) with or without Asfotase alfa (0.1-10 microg/mL) for 14-21 days. Mineralization is assessed by Alizarin Red S (ARS) or von Kossa staining to quantify calcium deposits. Alternatively, the hydrolysis of pyrophosphate (PPi) in the culture medium can be measured by an enzymatic method, and the expression of bone mineralization markers (e.g., osteocalcin, Osterix, Runx2) is measured by qPCR.
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| Animal Protocol |
Animal/Disease Models: Akp2−/− knockout mice[1]
Doses: 0.5, 2.0 and 8.2 mg/kg Route of Administration: subcutaneous (sc) injection; one time/day for 43 days Experimental Results: Dose-dependently increased the survival rate of mice, bone mineralization and prevention of mineralization defects of the feet, rib cage, lower limbs, jaw bones, the median survival, body weight, and bone length. demonstrated a clear relationship between daily dose and achieving a healthy BW and the ED80 (the dose that prevents bone defects in 80% of mice) was 3.2, 2.8 and 2.9 mg/kg/day for these sites. Remained increasing urinary PPi concentrations in all treatment groups. The in vivo efficacy of Asfotase alfa is assessed in the Akp2-/- knockout mouse model of infantile hypophosphatasia. Newborn Akp2-/- mice develop severe skeletal abnormalities, seizures, and die within 14 days. Treatment is initiated at birth or within 24 hours after birth. Asfotase alfa is administered at doses of 0.5, 2.0, and 8.2 mg/kg via subcutaneous injection once daily for 43 days. Endpoints include survival rate, median survival time, body weight, femur length (measured by X-ray or calipers), and bone mineralization measured by micro-CT (bone volume/total volume, trabecular thickness, bone mineral density). Histological staining of bone sections with von Kossa or Goldner's trichrome is performed to assess the extent of mineralization. |
| ADME/Pharmacokinetics |
Asfotase alfa is a large glycoprotein (molecular weight ~161,123 Da) with a formula of C7108H11008N1968O2206S56. It is administered as a subcutaneous (SC) injection. In humans, the plasma half-life ranges from 5 to 20 days, depending on age and dosing regimen. In Akp2-/- mice, once-daily SC dosing of 8.2 mg/kg for 43 days is used. The drug is designed to be bone-targeted, leading to prolonged retention at the site of action. In vitro, the compound is stable under recommended storage conditions (2-8degC) and should not be frozen.
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| Toxicity/Toxicokinetics |
Asfotase alfa has been evaluated in clinical trials and post-marketing surveillance for HPP. The most common adverse reactions in clinical trials (incidence ≥10%) included injection site reactions (e.g., bruising, pain, swelling, erythema), pyrexia (fever), headache, vomiting, hypophosphatemia, and paresthesia. Severe hypersensitivity reactions (anaphylaxis) have been reported in some patients. Lipodystrophy (localized fat loss or fat accumulation) at the injection site has also been observed. In animal studies, the drug is generally well-tolerated, with no significant systemic toxicity reported at therapeutic doses. The drug is administered by healthcare professionals.
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| References |
[1]. Yadav MC, et al. Dose response of bone-targeted enzyme replacement for murine hypophosphatasia. Bone. 2011 Aug;49(2):250-6.
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| Additional Infomation |
Asfotase alfa (ENB-0040) is approved under the brand name Strensiq (Alexion Pharmaceuticals) for the treatment of patients with perinatal, infantile, and juvenile-onset hypophosphatasia (HPP). It was granted breakthrough therapy designation by the FDA and approved in the US in 2015, and in the EU in 2016. It is a first-in-class bone-targeted enzyme replacement therapy designed to address the underlying cause of hypophosphatasia-deficient alkaline phosphatase (ALP). This drug is widely used in clinical practice for HPP and has significantly improved the prognosis for affected infants and children. The CAS registry number is 1174277-80-5.
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| CAS # |
1174277-80-5
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| Appearance |
Typically exists as solid at room temperature
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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