PEGylated cystathionine beta synthase for enzyme therapy for treatment of homocystinuria

A technology for synthesizing enzymes and cystathionine, which is applied in the direction of enzymes, lyases, and drug combinations, and can solve problems such as limitations, anxiety about long-term medical consequences of diseases, etc.

Pending Publication Date: 2022-07-22
特拉维尔治疗瑞士有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although no studies have been published on the quality of life (QoL) of patients with CBSDH, it has been observed that patients and their caregivers suffer psychosocial effects from following and administering a highly restricted and socially isolated diet, and are extremely anxious about the long-term medical consequences of the disease

Method used

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  • PEGylated cystathionine beta synthase for enzyme therapy for treatment of homocystinuria
  • PEGylated cystathionine beta synthase for enzyme therapy for treatment of homocystinuria
  • PEGylated cystathionine beta synthase for enzyme therapy for treatment of homocystinuria

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Experimental program
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Effect test

Embodiment 1

[0375] Example 1. Experimental protocol

[0376] A. Fermentation

[0377] Expression vectors with sequences encoding truncated human CBS were transformed into B1-21(DE3) E. coli bacteria, and bacteria from kanamycin resistant clones were grown in cells with 30 μg / mL kanamycin. Grow in 5 ml Luria-Bertani (LB) medium overnight at 37°C on a rotary shaker at 275 rpm. 1 mL of the overnight culture was added to 100 mL of TB broth (Terrific Broth) medium with 30 μg / mL kanamycin and grown overnight. 10 ml of the preculture was then added to 1 liter of TB medium containing 0.001% thiamine-HCl pH 8.0, 0.0025% pyridoxine-HCl pH 8.0, 0.3 mM delta-ALA pH 8.0, 150 μM chloride iron, 30 μg / mL kanamycin. Cultures were then grown at 30°C on a rotary shaker at 275 rpm until OD600 reached a value of about 0.6-0.7, and protein expression was induced by addition of 1 mM IPTG. Continue to ferment for an additional 16 hours. Cells were harvested by centrifugation at 6000 relative centrifugal f...

Embodiment 2

[0423] Example 2. Measurement of Reduction in PEGylation Levels in Pharmaceutical Formulations

[0424] Production of drug product formulations with varying degrees of dePEGylation for accelerated stability studies by incubating drug products at 25°C for two days, one month or six months under "Good Manufacturing Practice" (GMP) conditions for drug products to proceed. Store control samples within the recommended temperature range, i.e. below -65°C.

[0425] Differences in PEGylation between formulations were confirmed by reverse phase high performance liquid chromatography (RP-HPLC). Ten highly PEGylated and less PEGylated species were distinguished, as well as CBS without PEGylation and the heme cofactor (P10) released after enzymatic denaturation. The degree of dePEGylation in each sample was determined by comparing the relative areas of peaks corresponding to variably PEGylated or fully dePEGylated species. The RP-HPLC results are shown in Table 5. "CBS" is the nativ...

Embodiment 3

[0430] Example 3. Efficacy of formulations with reduced levels of PEGylation in mice

[0431] To assess bioequivalence of the drug product and its dePEGylated formulation, efficacy was analyzed in I278T CBS- / -(I278T- / -) mice, a model that reproduces the biochemical sequelae of CBSDH, namely Abnormal plasma levels of methionine (Met) metabolites, including elevation of homocysteine ​​(Hcy) and inhibition of cysteine ​​(Cys). Plasma aminothiols (total Cys and Hcy) and residual amino acids as well as SAM and SAH were measured by LC-MS / MS as described in: Arning et al. (2016) Methods Mol Biol 1378, 255-262, which are incorporated herein by reference in their entirety.

[0432] I278T CBS- / - (I278T- / -) mice lack the mouse CBS gene and express the I278T mutant human CBS gene carrying the most widely found pathogenic mutation in CBSDH patients. These mice express about 2%-3% of wild-type CBS activity and have a homocystinuric (HCU) phenotype (see Wang, et al. (2005) Human Molecula...

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Abstract

The present disclosure provides a formulation of a drug comprising a PEGylated CBS protein having the amino acid sequence of SEQ ID NO: 1. Doses and dosing regimens for treating homocystinuria in a subject in need thereof are provided. In addition, doses and dosing regimens for reducing homocysteine (Hcy) levels or increasing cysteine (Cys) and / or cystathionine (Cth) levels in a subject in need thereof are also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS [0002] This application claims 62 / 866,810, entitled Drug Product For Enzyme Replacement Therapy for Treatment of Homocystinuria, filed June 26, 2019 and March 2, 2020 Priority of application 62 / 983,860 entitled Drug Product For Enzyme Therapy for Treatment of Homocystinuria, the contents of each of which is incorporated by reference in its entirety manner is incorporated herein. [0003] Reference to Sequence Listing [0004] This application is filed with the Sequence Listing in electronic format. The Sequence Listing file titled 2089_1005PCT_SL.txt was created on June 26, 2020 and is 18,401 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety. [0005] Field of Invention [0006] The present disclosure relates to compositions and methods of enzyme therapy for treating homocystinuria using the pharmaceutical products described herein. Backgro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/51A61K47/60A61K47/26A61K9/19A61P3/00A61P27/02A61P1/08A61P9/00A61P25/00
CPCA61K38/51A61K47/60A61K47/26A61K9/19A61P3/00A61P27/02A61P1/08A61P9/00A61P25/00C12Y402/01022A61K47/02A61K45/06A61P43/00
Inventor E·布布利尔F·格拉文M·塞洛斯-莫拉T·马伊坦J·P·克劳斯R·万纳O·考斯维克
Owner 特拉维尔治疗瑞士有限公司
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