Methods for treating farber disease

Abstract

To disclose methods of treating Farber disease using particular doses and pharmacokinetic profiles.SOLUTION: A method of treating Farber disease in a human subject in need thereof is provided, the method comprising administering to the human subject purified recombinant human acid ceramidase (rhAC) in activated form at a dose of about 1 mg / kg to about 10 mg / kg. In the method, the dose may be about 1 mg / kg to about 5 mg / kg. In the method, the dose may be about 1 mg / kg.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 62 / 625,763, filed February 2, 2018, the subject matter of which is incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on January 17, 2019, is named RVT-801-006WO_SL.txt and is 11,363 bytes in size. [Background technology]

[0003] Farber disease, a lysosomal storage disorder (LSD), was first described in 1952 in a 14-month-old infant with granulomatous lesions in multiple joints and evidence of lipid accumulation. Over the next decade, other similar cases were described, all of which showed similar lesions and often a characteristic "hoarse" cry or voice due to the presence of lesions in the larynx. Involvement of other organ systems, including the lungs, liver, spleen, and central nervous system (CNS), was also noted in some of these patients.

[0004] Previously, treatment for patients with Farber disease was symptomatic and aimed primarily at reducing pain. Hematopoietic stem cell transplantation (HSCT) has been performed in a limited number of patients, with generally good outcomes, provided that the transplant procedure itself is successful. Successfully transplanted patients show significant reduction in pain, increased motility and mobility, and in some cases, reduction and complete resolution of subcutaneous nodules. However, successful transplantation requires histocompatible donor cells, exposing patients to invasive and potentially dangerous immunosuppressive regimens. One alternative to HSCT is gene therapy, in which autologous donor cells are transduced with vectors expressing therapeutic proteins, eliminating the need for a histocompatible donor. This technique has been evaluated in knock-in mouse models of Farber disease, resulting in a reduction in tissue ceramide and infiltration of macrophages. However, improved treatments for treating Farber disease continue to be needed.

[0005] Previous studies of mouse models of severe Farber disease have established a broad therapeutic dose range of recombinant human acid ceramidase ("rhAC") and characterized efficacy based on reduction of accumulated tissue ceramide and reduction of inflammatory cytokines. International Application No. PCT / US18 / 13509, filed January 12, 2018 (published as WO2018 / 132667 on July 18, 2018) and He et al., 2017 (Feb.), "Enzyme replacement therapy for Farber disProof-of-concept studies in cells and mice," BBA Clin. 13(7):85-96, are incorporated herein by reference in their entireties.

[0006] However, these studies did not establish exposure targets. Predicting the human equivalent dose (HED) from the therapeutic dose determined during non-clinical trials in mouse models of severe Farber disease requires an understanding of the pharmacokinetics ("PK") and tissue distribution ("TD") of the enzyme. The present subject matter, as discussed herein, fulfills such a need. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2018 / 132667 Brochure [Non-patent literature]

[0008] [Non-Patent Document 1] “Enzyme replacement therapy for Farber disProof-of-concept studies in cells and mice,”BBA Clin.13(7):85-96 Summary of the Invention

[0009] Young, healthy CD-1 mice are considered the parental strain of the Farber mouse (the Farber disease mouse model used in this description and examples). Figure 3 shows that the pharmacokinetic profile of the young CD-1 mouse rhAC (RVT-801) is similar to the Farber mouse experimental rhAC activity profile in circulation. This indicates that young CD-1 mice provide a good approximation of Farber mouse PK, and therefore CD-1 mice were used to characterize the mouse pharmacokinetics of RVT-801, since Farber mice are fragile, difficult to breed, and not available in sufficient numbers to perform a thorough PK evaluation.

[0010] Thus, based on a small number of Ferber mice and with limited overlapping data points between strains, the data reported in the Description and Examples herein suggest that systemic rhAC (e.g., RVT-801) exposure in CD-1 mice approximates that in Ferber mice and may represent the minimum systemic level of rhAC in Ferber mice following intraperitoneal administration of a single dose of RVT-801.

[0011] In the present description and examples, recombinant human AC (e.g., RVT-801) is shown to be widely distributed in tissues related to ceramide accumulation in Farber disease mouse models (e.g., liver, spleen, and lung).Therefore, measuring the whole-body level of recombinant human AC (e.g., RVT-801) may underestimate the exposure in the relevant tissues of human subjects suffering from Farber disease.Therefore, relying on simple allometry to scale doses in non-clinical species to humans may not be able to adequately estimate tissue exposure to provide efficacy.

[0012] Human equivalent dose (HED) estimates based on the U.S. Food and Drug Administration's ("FDA") guidance on scaling dosages between non-clinical species and humans are based on body surface area ("BSA") (USDept. of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), 2005, "Guidance for Industry. Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers." pp. 1-30), which is incorporated herein by reference in its entirety. However, the HED estimation approach described herein also takes into account physiological factors, including, but not limited to, the primary clearance mechanism from the vascular system to tissues.

[0013] The data reported here in mice indicate that the liver and spleen are the major target tissues for uptake of RVT-801 following systemic administration.

[0014] The HED may be derived by combining the BSA scaling and the ratio of tissue mass to body weight ("BW") in mice and humans. In this way, it is possible to obtain a dose scaling factor that accounts for the relative size of each compartment of recombinant human AC (RVT-801) distribution in each species. Combined HED estimates using the BSA and tissue:BW strategies are shown in Table 1. [Table 1]

[0015] In accordance with the present description and examples, in one embodiment, a method is provided for treating an adult human subject having Farber disease, comprising administering to the human subject a dose of about 0.8 mg / kg of recombinant human acid ceramidase (rhAC).

[0016] Also provided is a method of treating a human child with Farber disease, comprising administering to the child a dose of about 1.2 mg / kg of recombinant human acid ceramidase (rhAC), in one embodiment.

[0017] Also provided is a method of treating a human child with Farber disease, in one embodiment, comprising administering to the child a dose of about 2 mg / kg of recombinant human acid ceramidase (rhAC).

[0018] Also provided is a method of treating a human child with Farber disease, in one embodiment, comprising administering to the child a dose of about 5 mg / kg of recombinant human acid ceramidase (rhAC).

[0019] In some embodiments, a method of treating a human subject with Farber disease may comprise administering to the human subject a therapeutically effective dose of recombinant human acid ceramidase (rhAC), which may be determined based on a pharmacokinetic profile obtained in young, healthy CD-1 mice receiving a single, 10 mg / kg maximally effective dose ("MED") of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC exhibits a T of about 0.25 to 1.0 hours in healthy CD-1 mice, as disclosed herein. max , C of about 1.23 to about 2.17 μg / mL max , or an area under the curve (AUC) of about 1.37 h*μg / mL to about 1.49 h*μg / mL (or about 1 h*μg / mL to about 2 h*μg / mL). The maximum effective dose (MED) in Ferber mice was determined to be 10 mg / kg delivered by bolus IP injection. AUC determined after a single dose of 10 mg / kg in age-matched wild-type CD-1 mice was last The value of C is 1.37 μg.h / mL. max can be 1.23 μg / mL.

[0020] In some embodiments, a method of treating a human subject with Farber disease may comprise administering to the subject a therapeutically effective dose of rhAC, which may be determined based on a pharmacokinetic profile obtained in young, healthy CD-1 mice receiving a single, 10 mg / kg maximally effective dose ("MED") of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC exhibits an AUC 肝臓 :AUC 血清 , AUC 脾臓 :AUC 血清 , AUC 肺 :AUC 血清 , AUC 腎臓 :AUC 血清 , AUC 心臓 :AUC 血清Without being bound by theory, it is believed that the distribution of rhAC (RVT-801) to these tissues is important with respect to the overall efficacy of drug treatment. It is possible that glycosylation of RVT-801 plays a role in the uptake of the drug into these diseased tissues and thus positively impacts the efficacy of rhAC treatment.

[0021] In some embodiments, a method of treating a human subject with Farber disease may comprise administering to the subject a therapeutically effective dose of rhAC, which may be determined based on a pharmacokinetic profile obtained in young, healthy CD-1 mice receiving a single, 10 mg / kg maximally effective dose ("MED") of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC has an AUC of about 92.9 in healthy CD-1 mice. 肝臓 :AUC 血清 Ratio, AUC of about 47.6 脾臓 :AUC 血清 Ratio, AUC of about 5.64 肺 :AUC 血清 Ratio, AUC of about 17.3 腎臓 :AUC 血清 Ratio, AUC of about 2.69 心臓 :AUC 血清 Ratio, AUC of about 0.575 全血 :AUC 血清 Ratio, AUC of about 0.0281 脳 :AUC 血清 , or an AUC of approximately 0.000165 BALF :AUC 血清 In some embodiments, a method of treating a human subject with Farber disease may include administering to the subject a therapeutically effective dose of rhAC, which may be determined based on a pharmacokinetic profile obtained in young, healthy CD-1 mice receiving a single, 10 mg / kg maximally effective dose ("MED") of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC has an AUC 肝臓 :AUC 血清 , AUC 脾臓 :AUC 血清 , AUC肺 :AUC 血清 , AUC 腎臓 :AUC 血清 , AUC 心臓 :AUC 血清 This results in a ratio greater than 1 for

[0022] Without being bound by theory, it is believed that the distribution of rhAC (RVT-801) to these tissues is important with respect to the overall effectiveness of the drug treatment.

[0023] In some embodiments, a method of treating a human subject with Farber disease may include administering to the subject a therapeutically effective dose of rhAC, which may be determined based on a pharmacokinetic profile obtained in young, healthy CD-1 mice receiving a single, 10 mg / kg maximally effective dose ("MED") of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC has a T of about 0.25 to 1.0 hours in healthy CD-1 mice. max This results in:

[0024] In some embodiments, a method of treating a human subject with Farber disease may include administering to the subject a therapeutically effective dose of rhAC, which may be determined based on a pharmacokinetic profile obtained in young, healthy CD-1 mice receiving a single, 10 mg / kg maximally effective dose ("MED") of rhAC (i.e., RVT-801) intraperitoneally, where the rhAC has a C of about 1.23 μg / mL to 2.17 μg / mL in healthy CD-1 mice. max This results in:

[0025] In some embodiments, a method of treating a human subject with Farber disease may include administering to the subject a therapeutically effective dose of rhAC, which may be determined based on a pharmacokinetic profile obtained in young, healthy CD-1 mice that received a single, 10 mg / kg maximally effective dose ("MED") of rhAC (i.e., RVT-801) intraperitoneally, where the rhAC produces an AUC of about 1.37-1.49*μg / mL in healthy CD-1 mice.

[0026] In some embodiments, a method of treating a human subject with Farber disease may comprise administering to the human subject a therapeutically effective dose of rhAC, which may be determined based on the pharmacokinetic profile obtained in young, healthy CD-1 mice receiving a single, 10 mg / kg maximally effective dose ("MED") of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC induces C in tissues or matrices in healthy CD-1 mice according to the values ​​shown in FIG. max or AUC 0~last Or AUC 組織 / AUC 血清 This results in:

[0027] In some embodiments, a method of treating a human subject with Farber disease may include administering to the subject a therapeutically effective dose of rhAC, which may be determined based on a pharmacokinetic profile obtained in young, healthy CD-1 mice receiving a single, 10 mg / kg maximally effective dose ("MED") of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC has an AUC of about 138 hr*μg / mL in liver tissue in healthy CD-1 mice. 0~last This results in:

[0028] In some embodiments, a method of treating a human subject with Farber disease may include administering to the subject a therapeutically effective dose of rhAC, which may be determined based on a pharmacokinetic profile obtained in young, healthy CD-1 mice receiving a single, 10 mg / kg maximally effective dose ("MED") of rhAC (i.e., RVT-801) intraperitoneally, the rhAC having a C of 13 μg / mL of drug in liver tissue in healthy CD-1 mice. max This results in:

[0029] In some embodiments, a method of treating a human subject with Farber disease may comprise administering to the subject a therapeutically effective dose of rhAC, which may be determined based on a pharmacokinetic profile obtained in young, healthy CD-1 mice receiving a single, 10 mg / kg maximally effective dose ("MED") of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC exhibits an AUC of about 70.9 hr*μg / mL in spleen tissue in healthy CD-1 mice. 0~last This results in:

[0030] In some embodiments, a method of treating a human subject with Farber disease may comprise administering to the subject a therapeutically effective dose of rhAC, which may be determined based on a pharmacokinetic profile obtained in young, healthy CD-1 mice receiving a single, 10 mg / kg maximally effective dose ("MED") of rhAC (i.e., RVT-801) intraperitoneally, the rhAC having a C of about 7.58 μg / mL of drug in the spleen in healthy CD-1 mice. max This results in:

[0031] In some embodiments, a method of treating a human subject with Farber disease may include administering to the subject a therapeutically effective dose of rhAC, which may be determined based on a pharmacokinetic profile obtained in young, healthy CD-1 mice receiving a single, 10 mg / kg maximally effective dose ("MED") of rhAC (i.e., RVT-801) intraperitoneally, where the rhAC has an AUC of about 25.8 hr*μg / mL in kidney tissue in healthy CD-1 mice. 0~last This results in:

[0032] In some embodiments, a method of treating a human subject with Farber disease may include administering to the subject a therapeutically effective dose of rhAC (i.e., RVT-801), which may be determined based on a pharmacokinetic profile obtained in young, healthy CD-1 mice receiving a single, 10 mg / kg maximally effective dose ("MED") of rhAC intraperitoneally, the rhAC having a C of about 2.61 μg / mL of drug in kidney tissue in healthy CD-1 mice. max This results in:

[0033] In some embodiments, a method of treating a human subject with Farber disease may include administering to the subject a therapeutically effective dose of rhAC, which may be determined based on a pharmacokinetic profile obtained in young, healthy CD-1 mice receiving a single, 10 mg / kg maximally effective dose ("MED") of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC has an AUC of about 4.01 hr*μg / mL in cardiac tissue in healthy CD-1 mice. 0~last This results in:

[0034] In some embodiments, a method of treating a human subject with Farber disease may include administering to the subject a therapeutically effective dose of rhAC, which may be determined based on a pharmacokinetic profile obtained in young, healthy CD-1 mice receiving a single, 10 mg / kg maximally effective dose ("MED") of rhAC (i.e., RVT-801) intraperitoneally, the rhAC having a C of about 0.362 μg / mL of drug in cardiac tissue in healthy CD-1 mice. max This results in:

[0035] In some embodiments, a method of treating a human subject with Farber disease may include administering to the subject a therapeutically effective dose of rhAC, which may be determined based on a pharmacokinetic profile obtained in young, healthy CD-1 mice that received a single 10 mg / kg maximally effective dose ("MED") of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC has an AUC of about 0.0419 hr*μg / mL in brain tissue in healthy CD-1 mice. 0~last This results in:

[0036] In some embodiments, a method of treating a human subject with Farber disease may include administering to the subject a therapeutically effective dose of rhAC, which may be determined based on a pharmacokinetic profile obtained in young, healthy CD-1 mice receiving a single 10 mg / kg maximally effective dose ("MED") of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC induces a C of about 0.147 μg / mL in the brain in healthy CD-1 mice. max This results in:

[0037] In some embodiments, a method of treating a human subject with Farber disease may include administering to the subject a therapeutically effective dose of rhAC, which may be determined based on a pharmacokinetic profile obtained in young, healthy CD-1 mice receiving a single, 10 mg / kg maximally effective dose ("MED") of rhAC (i.e., RVT-801) intraperitoneally, where the rhAC has an AUC in blood of about 0.858 hr*μg / mL in healthy CD-1 mice. 0~last This results in:

[0038] In some embodiments, a method of treating a human subject with Farber disease may comprise administering to the subject a therapeutically effective dose of rhAC, which may be determined based on a pharmacokinetic profile obtained in young, healthy CD-1 mice receiving a single 10 mg / kg maximally effective dose ("MED") of rhAC (i.e., RVT-801) intraperitoneally, the rhAC having a C of about 1.23 μg / mL of drug in the brain in healthy CD-1 mice. max This results in:

[0039] In some embodiments, a method of treating a human subject with Farber disease may comprise administering to the human subject a therapeutically effective dose of recombinant human acid ceramidase (rhAC), which may be determined based on a pharmacokinetic profile obtained in young, healthy CD-1 mice receiving a single, 10 mg / kg maximally effective dose ("MED") of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC has an AUC of about 0.000245 hr*μg / mL in the BALF. 0~last to provide.

[0040] In some embodiments, a method of treating a human subject with Farber disease may include administering to the subject a therapeutically effective dose of rhAC, which may be determined based on a pharmacokinetic profile obtained in young, healthy CD-1 mice receiving a single 10 mg / kg maximally effective dose ("MED") of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC has a C for drug in BALF of about 0.00196 μg / mL in healthy CD-1 mice. max This results in:

[0041] In some embodiments, a method of treating a human subject with Farber disease may include administering to the subject a therapeutically effective dose of rhAC, which may be determined based on a pharmacokinetic profile obtained in young, healthy CD-1 mice receiving a single, 10 mg / kg maximally effective dose ("MED") of rhAC (i.e., RVT-801) intraperitoneally, where the rhAC has an AUC of about 8.4 hr*μg / mL in lung tissue in healthy CD-1 mice. 0~last This results in:

[0042] In some embodiments, a method of treating a human subject with Farber disease may include administering to the subject a therapeutically effective dose of rhAC, which may be determined based on a pharmacokinetic profile obtained in young, healthy CD-1 mice receiving a single 10 mg / kg maximally effective dose ("MED") of rhAC (i.e., RVT-801) intraperitoneally, the rhAC having a C of about 1.42 μg / mL of drug in lung tissue in healthy CD-1 mice. max This results in:

[0043] Additional objects and advantages will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0044] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the scope of the appended claims.

[0045] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one or more embodiment(s) and, together with the description, serve to explain the principles described herein. [Brief description of the drawings]

[0046] [Figure 1] FIG. 1 shows the time course of serum RVT-801 levels in RVT-801-treated Farber disease mice and age-matched healthy young CD-1 mice following a single injection of RVT-801 at doses of 1, 3, and 10 mg / kg according to Example 1. [Diagram 2] According to Example 1, pharmacokinetic metrics of serum RVT-801 in healthy juvenile CD-1 mice following a single injection of RVT-801 at doses of 1, 3 and 10 mg / kg are listed. [Diagram 3] According to Example 1, RVT-801 concentrations in serum of healthy age-matched CD-1 mice and rhAC activity in plasma of Farber disease mice following a single injection of RVT-801 at a dose of 10 mg / kg are shown, demonstrating comparability between the pharmacokinetic profiles of Farber mice and age-matched parental CD-1 strain mice. [Figure 4] According to Example 2, the time course of RVT-801 concentration in serum and various tissues (liver, spleen, kidney, heart, lung, and brain) of healthy young CD-1 mice after a single injection of serum RVT-801 at a dose of 10 mg / kg is shown. [Figure 5A] 1 shows the time course of RVT-801 concentration in liver tissue of healthy young CD-1 mice following a single injection of RVT-801 at doses of 1, 3, and 10 mg / kg according to Example 2. [Figure 5B]1 shows the time course of RVT-801 concentrations in spleen tissue of healthy young CD-1 mice following a single injection of RVT-801 at doses of 1, 3, and 10 mg / kg according to Example 2. [Figure 6A] According to Example 2, the AUC0-last in serum, whole blood, liver tissue extracts, and spleen tissue of healthy young CD-1 mice after a single injection of RVT-801 at doses of 1, 3, and 10 mg / kg is compared. [Figure 6B] FIG. 1 shows the AUC tissue / AUC serum AUC ratio calculated from RVT-801 concentrations in serum and liver and spleen tissues of healthy young CD-1 mice after a single injection of RVT-801 at doses of 1, 3, and 10 mg / kg according to Example 2. [Figure 7] According to Example 2, Cmax, AUC0-last, and AUC tissue / AUC serum calculated from RVT-801 concentrations in serum, whole blood, and various tissue extracts (liver, spleen, kidney, lung, heart, brain, and bronchoalveolar lavage fluid (BALF)) of healthy juvenile CD-1 mice after a single injection of RVT-801 at a dose of 10 mg / kg are shown. [Figure 8] FIG. 1 shows tissue distribution and comparison of AUC tissue / AUC serum in various tissues of healthy young CD-1 mice after a single injection of RVT-801 at a dose of 10 mg / kg according to Example 2. [Figure 9] 1 shows the estimated human equivalent dose (HED) scaled by body surface area (BSA) estimated from the MED of 10 mg / kg for reduction of accumulated ceramide in the liver and spleen of Ferber mice according to Example 3. [Figure 10] According to Example 3, tissue-specific HED scaled by tissue-to-body weight ratios estimated from a MED of 10 mg / kg in Faber mice for reduction of accumulated ceramide in the liver and spleen are shown. [Figure 11] According to Example 3, results are shown for both (1) a BSA-based HED calculation and (2) a tissue-to-body weight ratio-based HED calculation. [Figure 12]1 shows a comparison of an embodiment of the HED of RVT-801 in the present application with the administration of approved enzyme replacement therapy (ERT). [Figure 13] Mean rhAC concentration-time data in liver, spleen, and kidney of Ferber mice following single or repeated weekly doses of 10 mg / kg RVT-801 are shown. [Figure 14] Data are presented on rhAC serum concentrations in healthy age-matched CD-1 mice and acid ceramidase plasma activity in Faber mice following a single 10 mg / kg dose of RVT-801. [Figure 15] 1 shows the final rhAC tissue:serum AUC ratio in healthy young CD-1 mice following a single 10 mg / kg dose of RVT-801. [Figure 16] Shows circulating rhAC following a single 10 mg / kg IP bolus injection of RVT-801 in CD-1 and Ferber mice. [Figure 17] Circulating and major tissue concentrations of rhAC are shown following a single 10 mg / kg bolus IP injection of RVT-801 in CD-1 and Ferber mice. [Figure 18A] Individual rhAC concentration-time data are shown in the liver of (Faber (open symbols) and age-matched CD-1 (filled symbols)) mice following a single 10 mg / kg bolus IP injection of RVT-801 (n=3 per time point). [Figure 18B] Individual rhAC concentration-time data are shown in the spleens of (Faber (open symbols) and age-matched CD-1 (filled symbols)) mice following a single 10 mg / kg bolus IP injection of RVT-801 (n=3 per time point). [Figure 18C] Individual rhAC concentration-time data are shown in kidneys of (Faber (open symbols) and age-matched CD-1 (filled symbols)) mice following a single 10 mg / kg bolus IP injection of RVT-801 (n=3 per time point). [Figure 18D]Individual rhAC concentration-time data are shown in the lungs of (Faber (open symbols) and age-matched CD-1 (filled symbols)) mice following a single 10 mg / kg bolus IP injection of RVT-801 (n=3 per time point). [Figure 18E] Individual rhAC concentration-time data are shown in hearts of (Faber (open symbols) and age-matched CD-1 (filled symbols)) mice following a single 10 mg / kg bolus IP injection of RVT-801 (n=3 per time point). [Figure 19A] FIG. 1 shows the mean rhAC concentrations in circulating tissues of Faber mice (open symbols) and age-matched CD-1 mice (filled symbols) after either a single 10 mg / kg dose of RVT-801 administered by bolus IP injection or multiple weekly doses at 10 mg / kg / dose according to Example 5. [Figure 19B] FIG. 1 shows the mean rhAC concentrations in liver tissue of Farber mice (open symbols) and age-matched CD-1 mice (filled symbols) after either a single 10 mg / kg dose of RVT-801 administered by bolus IP injection or multiple weekly doses at 10 mg / kg / dose according to Example 5. [Figure 19C] FIG. 1 shows the mean rhAC concentrations in spleen tissue of Faber mice (open symbols) and age-matched CD-1 mice (filled symbols) after either a single 10 mg / kg dose of RVT-801 administered by bolus IP injection or multiple weekly doses at 10 mg / kg / dose according to Example 5. [Figure 19D] FIG. 1 shows the mean rhAC concentrations in circulating kidney tissue of Faber mice (open symbols) and age-matched CD-1 mice (filled symbols) after either a single 10 mg / kg dose of RVT-801 administered by bolus IP injection or multiple weekly doses at 10 mg / kg / dose according to Example 5. [Figure 19E]FIG. 1 shows the mean rhAC concentrations in cardiac tissue of Faber mice (open symbols) and age-matched CD-1 mice (filled symbols) after either a single 10 mg / kg dose of RVT-801 administered by bolus IP injection or multiple weekly doses at 10 mg / kg / dose according to Example 5. [Figure 19F] FIG. 1 shows the mean rhAC concentration in lungs (tissues) of Ferber mice (open symbols) and age-matched CD-1 mice (filled symbols) after either a single 10 mg / kg dose of RVT-801 administered by bolus IP injection or multiple weekly doses at 10 mg / kg / dose according to Example 5. [Figure 19G] FIG. 1 shows the mean rhAC concentrations in the brain (tissue) of Ferber mice (open symbols) and age-matched CD-1 mice (filled symbols) after either a single 10 mg / kg dose of RVT-801 administered by bolus IP injection or multiple weekly doses at 10 mg / kg / dose according to Example 5. [Figure 20A] The mean rhAC tissue concentration-time profiles following IP administration to young CD-1 mice in RVT-801-9013 part B, respectively (linear plots). [Figure 20B] 13A-13D show mean rhAC tissue concentration-time profiles following IP administration to young CD-1 mice in RVT-801-9013 part B, respectively (log-linear plots). [Figure 21] Figure 1 shows RVT-801 tissue:serum exposure ratios based on AUClast in BALF, blood, brain, heart, kidney, liver, lung, and spleen following a single dose of 1 mg / kg, 3 mg / kg, and 10 mg / kg RVT-801 in young CD-1 mice. [Figure 22A] 1 shows dose-normalized AUC plotted against dose level for various tissues following IP administration of RVT-801 at doses of 1 mg / kg, 3 mg / kg, and 10 mg / kg to young CD-1 mice. [Figure 22B]1 shows dose-normalized AUC plotted against dose level for various tissues following IP administration of RVT-801 at doses of 1 mg / kg, 3 mg / kg, and 10 mg / kg to young CD-1 mice. [Figure 22C] 1 shows dose-normalized AUC plotted against dose level for various tissues following IP administration of RVT-801 at doses of 1 mg / kg, 3 mg / kg, and 10 mg / kg to young CD-1 mice. [Figure 22D] 1 shows dose-normalized AUC plotted against dose level for various tissues following IP administration of RVT-801 at doses of 1 mg / kg, 3 mg / kg, and 10 mg / kg to young CD-1 mice.

[0047] Array Description [Table 2] JPEG2025041867000004.jpg251154JPEG2025041867000005.jpg160168JPEG2025041867000006.jpg211168JPEG2025041867000007.jpg213168 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] As used herein, the terms "a" or "an" mean "at least one" or "one or more," unless the context clearly indicates otherwise.

[0049] As used herein, the term "about" means that the numerical value is approximate and that small variations will not significantly affect the practice of the disclosed embodiments. When numerical limitations are used, unless otherwise indicated by the context, "about" means that the numerical value can vary by ±10% and remain within the scope of the disclosed embodiments.

[0050] As used herein, "acid sphingomyelinase activity" or "ASM activity" refers to a related lipid hydrolase that tightly binds and co-purifies with AC (Bernardo, K., R. Hurwitz, T. Zenk, RJ Desnick, K. Ferlinz, EH Schuchman, and K. Sandhoff, 1995, "Purification, characterization, and biosynthesis of human acid ceramidase," J Biol Chem, 270:11098-11102).

[0051] As used herein, the term "animal" includes, but is not limited to, humans and non-human vertebrates, such as wild animals, domestic animals, and farm animals. Animals may also be referred to as "subjects."

[0052] The term "carrier" as used herein means a diluent, adjuvant, or excipient with which a compound is administered. Pharmaceutical carriers can be liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Pharmaceutical carriers can also be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizers, thickeners, lubricants, and colorants can be used.

[0053] As used herein, "children" refers to ages from newborn to 18 years of age.

[0054] As used herein, "comprising" (and any form of comprising, such as "comprise," "comprises," and "comprised"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unlisted elements or method steps. Additionally, it is understood that terms used in conjunction with the term "comprising" can be used in conjunction with the terms "consisting of" or "consisting essentially of."

[0055] As used herein, the term "contact" refers to bringing two elements together in an in vitro system or in vivo system.For example, "contacting" rhAC polypeptide with an individual, subject, or cell includes administering the polypeptide to an individual or patient, such as a human, as well as introducing a compound into the cell or sample containing purified preparations that contain the polypeptide.Furthermore, contacting can refer to transfecting or infecting cells with the nucleic acid molecule that codes for the polypeptide.

[0056] As used herein, "Farber mouse", "Farber mice", "Farber disease mice", and "Farber disease mouse" refer to a mouse model of severe Farber disease (Asah1 P361R / P31R6) stands for. The Farber mouse is a mouse model based on the genotype of a patient with severe Farber disease. As previously described (Alayoubi, AM, JC Wang, BCAu, S. Carpentier, V. Garcia, S. Dworski, S. El-Ghamrasni, KN Kirouac, MJ Exertier, ZJ Xiong, GG Prive, CM Simonaro, J. Casas, G. Fabrias, EH Schuchman, PVTurner, R. Hakem, T. Levade, and JAMedin, 2013, Systemic ceramide accumulation leads to severe and varied pathological consequences, EMBO Mol Med, 5:827-842), a single nucleotide Asah1 mutation was identified based on the genotype of a patient with severe Farber disease. P361R / P361R Knock-in mice homozygous for the mutation were derived from a mixed genetic background colony (W4 / 129Sv / CD-1) ("CD-1 mice") to establish a mouse model of severe Farber disease. Farber mice have been established with knock-in mice homozygous for a single nucleotide mutation in the Asah1 gene. P361R / P31R6 ) mice produce altered ACs that are unable to hydrolyze ceramides into their sphingosine and fatty acid components. These Faber mice are similar to wild-type (Asah1 WT / WT ) or heterozygous (Asah1 WT / P361R ) They display characteristics characteristic of clinical Farber disease, including bone destruction and intra-articular tissue morphology, the presence of lipid-laden macrophages, and systemic inflammation, along with significantly impaired growth rate and shortened life span compared to littermates.

[0057] An "effective amount" of enzyme delivered to a subject is an amount sufficient to improve the clinical course of Farber disease, where clinical improvement is measured by any of a variety of defined parameters well known to those of skill in the art.

[0058] As used herein, the terms "subject," "individual," or "patient" are used interchangeably and refer to any animal, including mammals such as mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses, or primates, such as humans.

[0059] As used herein, the phrase "in need of" means that a subject is identified as needing a particular method or treatment. In some embodiments, the identification may be by any diagnostic means. A subject may be in need of any of the methods and treatments described herein.

[0060] As used herein, the phrase "an integer from X to Y" means any integer, including the endpoints. For example, the phrase "an integer from 1 to 5" means 1, 2, 3, 4, or 5.

[0061] As used herein, the term "isolated" means that the compounds described herein are separated, such as by conventional techniques, from other components of either (a) a natural source such as a plant or cell, or (b) a synthetic organic chemical reaction mixture.

[0062] As used herein, the term "mammal" refers to a rodent (i.e., a mouse, rat, or guinea pig), monkey, cat, dog, cow, horse, pig, or human. In some embodiments, the mammal is a human.

[0063] As used herein, the phrase "pharmacologically acceptable" means compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals. In some embodiments, "pharmacologically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias, for use in animals, and more specifically, in humans.

[0064] As used herein, the term "serum" refers to any blood-derived matrix, including but not limited to "serum," "plasma," or "whole blood."

[0065] As used herein, the phrase "substantially isolated" means a compound that is at least partially or substantially separated from the environment in which it is formed or detected.

[0066] The phrase "therapeutically effective amount" as used herein means an amount of an active compound or pharmaceutical agent that produces a biological or medical response sought in a tissue, system, animal, individual, or human by a researcher, veterinarian, physician, or other clinician. The therapeutic effect depends on the disorder being treated or the biological effect desired. Thus, the therapeutic effect may be a reduction in the severity of symptoms associated with the disorder and / or inhibition of the progression of the disorder (partial or complete), or an improved treatment, cure, prevention, or elimination of the disorder or side effect effects. The amount required to produce a therapeutic response can be determined based on the age, health, size, and sex of the subject. The optimal amount can also be determined based on monitoring the subject's response to the treatment.

[0067] Any method known to those skilled in the art may be used to monitor the disease state and the effectiveness of treatment. Clinical monitors of disease state may include, but are not limited to, ceramide levels, body weight, joint length, inflammation, or any other clinical phenotype known to be associated with Farber disease.

[0068] As used herein, the terms "treat", "treated" or "treating" refer to both therapeutic treatments and prophylactic measures, the purpose of which is to inhibit (alleviate) an undesirable physiological condition, disorder, or disease, or to obtain a beneficial or desired clinical outcome. For example, beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms; reduction in the extent of a condition, disorder, or disease; a stable (i.e., not worsening) state of a condition, disorder, or disease state; a delayed onset or slowing of progression of a condition, disorder, or disease; alleviation or remission (either partial or total) of a condition, disorder, or disease state, whether detectable or undetectable; an improvement in at least one measurable physical parameter, not necessarily discernible by the patient; or an enhanced or improved condition, disorder, or disease. Thus, "treatment of Farber disease" or "treating Farber disease" refers to an activity that alleviates or improves either the primary events or secondary symptoms associated with Farber disease or other conditions described herein.

[0069] It is further to be understood that certain features, which are described in this specification in the context of separate embodiments, for clarity, can also be provided in combination in a single embodiment. Conversely, various features, which are described in the context of a single embodiment for brevity, can also be provided separately or in any suitable subcombination.

[0070] In some embodiments, a method of treating Farber disease is provided. In some embodiments, the subject is a subject in need thereof. In some embodiments, the subject in need thereof has been diagnosed with Farber disease. In some embodiments, the subject is also identified as having: 1) a subcutaneous nodule; 2) an acid ceramidase activity value in leukocytes, cultured skin fibroblasts, or other biological source (e.g., plasma) that is less than 30% of a control value; and / or 3) a nucleotide change in both alleles of the acid ceramidase gene (ASAH1) that indicates possible loss of function of the acid ceramidase protein via bioinformatics, gene expression studies, and / or other methods. In some embodiments, the method comprises administering to the subject a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase from about 0.1 mg / kg to about 50 mg / kg. In some embodiments, the method comprises administering to a human adult subject a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase of about 0.8 mg / kg. In some embodiments, the method comprises administering to a human child a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase of about 1.2 mg / kg. In some embodiments, the method includes administering to the subject a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase of about 1 mg / kg to about 5 mg / kg, hi some embodiments, the method includes administering to the subject a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase of about 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg.

[0071] In some embodiments, a method of reducing lipogranulomas in a subject having or suspected of having Farber disease is provided. In some embodiments, the subject is a subject in need thereof. In some embodiments, the method comprises administering to the subject a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase from about 0.1 mg / kg to about 50 mg / kg. In some embodiments, the method comprises administering to a human adult subject a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase from about 0.8 mg / kg. In some embodiments, the method comprises administering to a human child a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase from about 1.2 mg / kg. In some embodiments, the method comprises administering to the subject a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase from about 1 mg / kg to about 5 mg / kg. In some embodiments, the method comprises administering to the subject a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase from about 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg.

[0072] In some embodiments, a method of reducing absolute or normalized spleen weight in a subject having or suspected of having Farber disease is provided. In some embodiments, the subject is a subject in need thereof. In some embodiments, the method comprises administering to the subject a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase from about 0.1 mg / kg to about 50 mg / kg. In some embodiments, the method comprises administering to a human adult subject a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase from about 0.8 mg / kg. In some embodiments, the method comprises administering to a human child a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase from about 1.2 mg / kg. In some embodiments, the method comprises administering to a subject a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase from about 1 mg / kg to about 5 mg / kg. In some embodiments, the method includes administering to a subject a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase of about 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg.

[0073] In some embodiments, a method of reducing ceramide levels in a subject having or suspected of having Farber disease is provided. In some embodiments, the subject is a subject in need thereof. In some embodiments, the method comprises administering to the subject a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase from about 0.1 mg / kg to about 50 mg / kg. In some embodiments, the method comprises administering to a human adult subject a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase from about 0.8 mg / kg. In some embodiments, the method comprises administering to a human child a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase from about 1.2 mg / kg. In some embodiments, the method comprises administering to the subject a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase from about 1 mg / kg to about 5 mg / kg. In some embodiments, the method comprises administering to the subject a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase from about 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg.

[0074] Ceramide reduction can also refer to a decrease in ceramide or an increase in ceramide metabolism that results in decreased ceramide levels.

[0075] In some embodiments, a method of increasing sphingosine levels in a subject having or suspected of having Farber disease is provided. In some embodiments, the subject is a subject in need thereof. In some embodiments, the method comprises administering to the subject a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase from about 0.1 mg / kg to about 50 mg / kg. In some embodiments, the method comprises administering to a human adult subject a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase from about 0.8 mg / kg. In some embodiments, the method comprises administering to a human child a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase from about 1.2 mg / kg. In some embodiments, the method comprises administering to the subject a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase from about 1 mg / kg to about 5 mg / kg. In some embodiments, the method comprises administering to the subject a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase from about 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg.

[0076] Various pharmaceutical compositions are described herein and can be used based on patient and physician preferences. However, in some embodiments, the pharmaceutical composition is a solution. In some embodiments, the pharmaceutical composition comprises a cell-conditioned medium containing rhAC. As used herein, the term "cell-conditioned medium" refers to a cell culture medium used to culture cells expressing rhAC, where the protein is secreted into the medium, and the protein is then isolated or purified from the medium. In some embodiments, the medium is used to treat a subject. The medium can be applied to the skin of a subject, for example, to treat any of the conditions, symptoms, or disorders described herein.

[0077] In addition to the routes of administration described herein, in some embodiments, the pharmaceutical composition is administered by contacting the skin of a subject. In some embodiments, the administration is parenteral. In some embodiments, the administration comprises injecting the pharmaceutical composition into a subject. In some embodiments, the administration is intraperitoneal or intravenous injection. In some embodiments, the administration is oral administration.

[0078] In some embodiments, a method of treating Farber disease in a subject in need thereof is provided, the method comprising expressing recombinant human acid ceramidase (rhAC) in a cell, isolating the expressed rhAC from the cell, and administering to the subject a pharmaceutical composition comprising an effective amount of about 0.1 mg / kg to about 50 mg / kg of the isolated expressed rhAC. In some embodiments, the method comprises administering to a human adult subject a pharmaceutical composition comprising an effective amount of about 0.8 mg / kg of recombinant human acid ceramidase. In some embodiments, the method comprises administering to a human child a pharmaceutical composition comprising an effective amount of about 1.2 mg / kg of recombinant human acid ceramidase. In some embodiments, the method comprises administering to a subject a pharmaceutical composition comprising an effective amount of about 1 mg / kg to about 5 mg / kg of recombinant human acid ceramidase. In some embodiments, the method includes administering to a subject a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase of about 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg.

[0079] In some embodiments, a method of reducing lipogranulomas in a subject having or suspected of having Farber disease is provided, the method comprising expressing recombinant human acid ceramidase (rhAC) in a cell, isolating the expressed rhAC from the cell, and administering to the subject a pharmaceutical composition comprising an effective amount of about 0.1 mg / kg to about 50 mg / kg of the isolated expressed rhAC. In some embodiments, the method comprises administering to a human adult subject a pharmaceutical composition comprising an effective amount of about 0.8 mg / kg of recombinant human acid ceramidase. In some embodiments, the method comprises administering to a human child a pharmaceutical composition comprising an effective amount of about 1.2 mg / kg of recombinant human acid ceramidase. In some embodiments, the method comprises administering to a subject a pharmaceutical composition comprising an effective amount of about 1 mg / kg to about 5 mg / kg of recombinant human acid ceramidase. In some embodiments, the method includes administering to a subject a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase of about 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg.

[0080] In some embodiments, a method is provided that includes treating lipogranulomatosis (now Farber disease) in a subject having or suspected of having Farber disease, the method includes expressing recombinant human acid ceramidase (rhAC) in a cell, isolating the expressed rhAC from the cell, and administering to the subject a pharmaceutical composition comprising an effective amount of about 0.1 mg / kg to about 50 mg / kg of the isolated expressed rhAC. In some embodiments, the method includes administering to a human adult subject a pharmaceutical composition comprising an effective amount of about 0.8 mg / kg of recombinant human acid ceramidase. In some embodiments, the method includes administering to a human child a pharmaceutical composition comprising an effective amount of about 1.2 mg / kg of recombinant human acid ceramidase. In some embodiments, the method includes administering to a subject a pharmaceutical composition comprising an effective amount of about 1 mg / kg to about 5 mg / kg of recombinant human acid ceramidase. In some embodiments, the method includes administering to a subject a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase of about 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg.

[0081] In some embodiments, a method is provided for reducing spleen weight (absolute or normalized to body weight) in a subject having or suspected of having Farber disease, the method comprising expressing recombinant human acid ceramidase (rhAC) in a cell, isolating the expressed rhAC from the cell, and administering to the subject a pharmaceutical composition comprising an effective amount of about 0.1 mg / kg to about 50 mg / kg of the isolated expressed rhAC. In some embodiments, the method comprises administering to a human adult subject a pharmaceutical composition comprising an effective amount of about 0.8 mg / kg of recombinant human acid ceramidase. In some embodiments, the method comprises administering to a human child a pharmaceutical composition comprising an effective amount of about 1.2 mg / kg of recombinant human acid ceramidase. In some embodiments, the method comprises administering to a subject a pharmaceutical composition comprising an effective amount of about 1 mg / kg to about 5 mg / kg of recombinant human acid ceramidase. In some embodiments, the method includes administering to a subject a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase of about 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg.

[0082] In some embodiments, a method of lowering ceramide in a subject having or suspected of having Farber disease is provided, the method comprising expressing recombinant human acid ceramidase (rhAC) in a cell, isolating the expressed rhAC from the cell, and administering to the subject a pharmaceutical composition comprising an effective amount of about 0.1 mg / kg to about 50 mg / kg of the isolated expressed rhAC. In some embodiments, the method comprises administering to a human adult subject a pharmaceutical composition comprising an effective amount of about 0.8 mg / kg of recombinant human acid ceramidase. In some embodiments, the method comprises administering to a human child a pharmaceutical composition comprising an effective amount of about 1.2 mg / kg of recombinant human acid ceramidase. In some embodiments, the method comprises administering to a subject a pharmaceutical composition comprising an effective amount of about 1 mg / kg to about 5 mg / kg of recombinant human acid ceramidase. In some embodiments, the method includes administering to a subject a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase of about 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg.

[0083] In some embodiments, a method for increasing sphingosine in a subject having or suspected of having Farber disease comprises expressing recombinant human acid ceramidase (rhAC) in a cell, isolating the expressed rhAC from the cell, and administering to the subject a pharmaceutical composition comprising an effective amount of about 0.1 mg / kg to about 50 mg / kg of the isolated expressed rhAC. In some embodiments, the method comprises administering to a human adult subject a pharmaceutical composition comprising an effective amount of about 0.8 mg / kg of recombinant human acid ceramidase. In some embodiments, the method comprises administering to a human child a pharmaceutical composition comprising an effective amount of about 1.2 mg / kg of recombinant human acid ceramidase. In some embodiments, the method comprises administering to a subject a pharmaceutical composition comprising an effective amount of about 1 mg / kg to about 5 mg / kg of recombinant human acid ceramidase. In some embodiments, the method includes administering to a subject a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase of about 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg.

[0084] In some embodiments, expressing recombinant human acid ceramidase (rhAC) in a cell comprises introducing a vector encoding rhAC into the cell. In some embodiments, the vector comprises a nucleic acid molecule encoding rhAC. In some embodiments, the nucleic acid molecule is a molecule described herein or any other nucleic acid molecule encoding a rhAC polypeptide or a homolog thereof as described in more detail herein. In some embodiments, the vector is a viral vector. For example, the vector can be a retroviral vector or a DNA viral vector such as adenovirus, AAV, etc. In some embodiments, the vector is a plasmid. In some embodiments, the vector comprises a promoter operably linked to rhAC. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an SV40 promoter, a CMV promoter, an EF1α promoter, or any combination thereof, or any other promoter active in mammalian cells.

[0085] In some embodiments, the vector transfects or infects the cell. The method of introducing the vector into the cell is not critical, and any method can be used that provides sufficient expression of the rhAC polypeptide in the cell.

[0086] In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is not a human cell. In some embodiments, the cell is a hamster cell. In some embodiments, the cell is a Chinese Hamster Ovary (CHO) cell. In some embodiments, the cell can be grown in a serum-free or substantially serum-free environment. In some embodiments, the cell is derived from a CHO-K1 cell. In some embodiments, the cell is a mouse cell. In some embodiments, the cell is a mouse myeloma cell. In some embodiments, the cell is an NS0 cell. In some embodiments, the effective amount administered is as described herein above and below.

[0087] In some embodiments, the pharmaceutical composition is administered as described herein. For example, in some embodiments, the composition is administered to a subject orally, by inhalation, intranasal injection, topically, transdermally, parenterally, subcutaneously, intravenously, intraarterially, intramuscularly, intrathoracically, intraperitoneally, intrathecally, or by application to a mucosa.

[0088] As used herein, the term "rhAC" or "AC" refers to recombinant human acid ceramidase encoded by the ASAH1 gene (NCBI UniGene GeneID No. 427). AC hydrolyzes the amide bond that connects the sphingosine and fatty acid moieties of lipid ceramide (Park, J.-H., Schuchman EH, 2006, "Acid ceramidase and human disease," Biochim. Biophys. Acta. 1758(12): 2133-2138; Nikolova-Karakashian et al., 2000, "Ceramidases," Methods Enzymol. 311: 194-201 (2000); Hassler et al., 1993, "Ceramidase: Enzymology and metabolic roles," Adv. Lipid Res. 26: 49-57). Mutations in both ASAH1 alleles can cause Farber disease.

[0089] There are three types of ceramidases described to date (Nikolova-Karakashian, 2000). These are classified as acidic, neutral, and alkaline ceramidases according to the optimum pH of enzymatic activity. AC has optimal enzymatic activity at pH<5. Human AC was the first ceramidase to be cloned (Koch et al., 1996, "Molecular Cloning and Characterization of a Full-length Complementary DNA Encoding Human Acid Ceramidase: Identification Of The First Molecular Lesion Causing Farber Disease," J. Biol. Chem. 271:33110-33115). It is localized in lysosomes and is mainly involved in the catabolism of ceramide. Malfunction of this enzyme due to a genetic defect results in a sphingolipidosis called lipogranulomatosis or Farber disease (Koch et al., 1996, "Molecular Cloning and Characterization of a Full-length Complementary DNA Encoding Human Acid Ceramidase: Identification Of The First Molecular Lesion Causing Farber Disease," J. Biol. Chem. 271:33110-33115; Young et al., 2013, "Sphingolipids: regulators of crosstalk between apotosis and autophagy," J. Lipid. Res. 54:5-19).

[0090] AC (N-acylsphingosine deacylase, IUBMB Enzyme No. EC 3.5.1.23) protein has been purified from several sources and human and mouse cDNAs and genes have been obtained. See Bernardo et al., J. Biol. Chem. 270:11098-102 (1995); Koch et al., J. Biol. Chem. 2711:33110-5 (1996); Li et al., Genomics 50:267-74 (1998); Li et al., Genomics 62:223-31 (1999). It is generated via cleavage of the AC precursor protein, the product of the ASAH1 gene (NCBI UniGene GeneID No. 427) (see Ferlinz et al., J. Biol. Chem. 276(38):35352-60 (2001)). The AC protein [Homo sapiens] (NCBI accession number AAC50907) is shown in SEQ ID NO:1.

[0091] The α subunit of AC begins at amino acid 22 and continues to amino acid 142 (shown in bold in SEQ ID NO:1 of the sequence listing). The β subunit of AC begins at amino acid 143 and continues to amino acid 395 (shown in italics in SEQ ID NO:1).

[0092] As used herein, "active acid ceramidase", "active AC" or "activated form of AC", or similar phrases refer to AC precursor protein that has undergone autoproteolytic cleavage into an active form (consisting of α- and β-subunits). The mechanism of cleavage and activation of human AC has been reported in (Shtraizent, N., E. Eliyahu, JH Park, X. He, R. Shalgi and EH Schuchman, 2008, "Autoproteolytic cleavage and activation of human acid ceramidase," J Biol Chem, 283(17):11253-11259). Activation is also driven by the intracellular environment and is expected to occur in most, if not all, cell types based on the highly conserved sequence at the cleavage site of the ceramidase precursor protein across species.

[0093] As used herein, "inactive acid ceramidase", "inactive AC", or "inactive acid ceramidase precursor", "inactive AC precursor", or (AC preprotein) refers to an AC precursor protein that has not undergone autoproteolytic cleavage to an active form. Inactive AC precursors and active ACs suitable for use in the recombinant acid ceramidase of this and all embodiments of the present invention can be homologous (i.e., from the same species) or heterologous (i.e., from a different species) to the tissue, cell, and / or subject being treated. The acid ceramidase (e.g., AC) precursor protein is autoproteolytically cleaved to its active form (consisting of α and β subunits). The mechanism of human AC cleavage and activation has been reported in (Shtraizent, 2008). Activation is also driven by the intracellular environment and is expected to occur in most, if not all, cell types based on the highly conserved sequence at the cleavage site of the ceramidase precursor protein across species. Thus, ceramidase as used herein includes both active ceramidase and ceramidase precursor protein, which is converted to active ceramidase protein by autoproteolytic cleavage. Both embodiments are contemplated in which the precursor protein is taken up by the cell of interest and thereby converted to active ceramidase, and in which the precursor protein is converted to active ceramidase by a different cell or agent (e.g., present in culture).

[0094] Active AC and inactive AC precursor proteins that can be used in this and all aspects of the invention include, but are not limited to, those listed in Table 1 of US 2016 / 0038574, the contents of which are incorporated herein by reference in their entirety. [Table 3]

[0095] In some embodiments, the rhAC comprises the amino acid sequence of SEQ ID NO:1.

[0096] "RVT-801" is an activated form of recombinant human acid ceramidase (rhAC) for the treatment of Farber disease. The α and β subunits of activated rhAC are linked by disulfide bonds. The molecule is recombinant human acid ceramidase (rhAC) derived from CHO-M cells transfected with a DNA plasmid vector expressing rhAC. RVT-801 is based on the UniProtKB code: Q13510.

[0097] The therapeutic efficacy of RVT-801rhAC has been established in a mouse model of severe Farber disease (He, et al., 2017) and characterized in multiple studies, with endpoints describing positive effects on histopathological and immunological outcomes, accompanied by a reduction in accumulated ceramide.

[0098] In some embodiments, the rhAC is a protein that is a homolog of SEQ ID NO:1. In some embodiments, the rhAC is encoded by a nucleic acid molecule of SEQ ID NO:2. In some embodiments, the rhAC is encoded by a nucleic acid molecule of SEQ ID NO:3. In some embodiments, the rhAC is encoded by a nucleic acid molecule of SEQ ID NO:4. In some embodiments, the sequence is as defined in GenBank Accession No. NM_177924.3 or NM_177924.4, each of which is incorporated by reference in its entirety. The nucleotide sequence encoding the protein can be the complete sequence shown in SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, or simply the coding region of the sequence. The coding region can be, for example, nucleotides 313-1500 of SEQ ID NO:2, or the corresponding coding region found in SEQ ID NO:3 or SEQ ID NO:4. However, as is well known to those of skill in the art, the genetic code is degenerate, and thus other codons can be used to encode the same protein without falling outside the scope of what is disclosed. Since the amino acid sequence is known, any nucleotide sequence that encodes the amino acid sequence is acceptable. In some embodiments, the nucleotide sequence includes a signal peptide. In some embodiments, the signal peptide is an amino acid sequence encoded by nucleotides 313-375 of SEQ ID NO:2. In some embodiments, the protein produced comprises a signal peptide of amino acid residues 1-21 of SEQ ID NO:1. In some embodiments, the protein produced does not include a signal peptide, such as the signal peptide of amino acid residues 1-21 of SEQ ID NO:1. In some embodiments, the signal peptide is removed during post-translational processing, where the enzyme is processed into different subunits. In some embodiments, the nucleotide sequence is codon optimized for the cell in which the protein is expressed. In some embodiments, the protein comprises an alpha subunit, a beta subunit, etc. In some embodiments, the protein produced comprises a peptide of amino acid residues 22-142, 45-139, 134-379, 143-395, or 1-395 of SEQ ID NO:1.The peptides can be polypeptides of different sequences to form a single protein or enzyme. In some embodiments, the protein does not include amino acid residues 1-21. These regions can be encoded by a single nucleotide sequence or separate nucleotide sequences or a combination of nucleotide sequences. As discussed herein, any nucleotide sequence that encodes a protein can be used and is not limited to the sequences set forth herein as SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

[0099] In some embodiments, the rhAC has acid ceramidase (AC) activity but no detectable acid sphingomyelinase activity, such as the rhAC produced in Examples 1 and 2 below. Acid sphingomyelinase activity may be removed, for example, by heat inactivation. Heat inactivation may remove other contaminating proteins from the rhAC preparation. See, for example, US Patent Publication No. 20160038574, which is incorporated herein in its entirety.

[0100] The term "homolog" refers to a protein sequence having 80% to 100% sequence identity to a reference sequence. The percent identity between two peptide chains can be determined by pair wise alignment using default settings in the AlignX module of Vector NTI v.9.0.0 (Invitrogen Corp., Carlsbad, Calif.). In some embodiments, a homolog has at least or about 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to a sequence described herein, e.g., SEQ ID NO:1.

[0101] In some embodiments, the protein delivered to the subject is a conservative substitution compared to the sequence described herein. Non-limiting exemplary conservative substitutions shown in Table 3 are included within the scope of the disclosed subject matter. Substitutions may be made to improve enzyme function, such as stability or enzyme activity. Conservative substitutions produce molecules that have similar functional and chemical characteristics to the molecule to which such modification is made. Exemplary amino acid substitutions are shown in Table 3 below. [Table 4]

[0102] As used herein, the term "in combination with" means that the recited agents can be administered to a subject together in a mixture either simultaneously as a single agent, or sequentially as a single agent in any order.

[0103] As described herein, in some embodiments, the protein is produced from a cell. In some embodiments, the cell is a Chinese Hamster Ovary cell, "CHO ​​cell". The nucleic acid sequence encoding the protein described herein can be genomic DNA or cDNA or RNA (e.g., mRNA) encoding at least one of the proteins described herein. The use of cDNA requires that the host cell's appropriate gene expression elements be combined with the gene to achieve synthesis of the desired protein. The use of cDNA sequences can be advantageous over genomic sequences (including introns) in that the cDNA sequences can be expressed in bacteria or other hosts that lack an appropriate RNA splicing system. Those skilled in the art can determine the best system for expressing the protein.

[0104] In some embodiments, the protein is produced according to U.S. Patent Application Publication No. 20160038574, which is incorporated by reference in its entirety.

[0105] Because the genetic code is degenerate, more than one codon can be used to code for a particular amino acid. Using the genetic code, one or more different oligonucleotides can be identified, each of which will be capable of coding for the amino acid sequence described herein.

[0106] The enzyme administered to a subject to treat Farber disease or a condition related thereto may be purified. The term "purified" with respect to a protein refers to a protein that is substantially free of other materials associated with the molecule in its natural environment. For example, a purified protein is substantially free of cellular material or other proteins from the cell or tissue from which the protein is derived. The term refers to an isolated protein that is sufficiently pure to be analyzed, or to preparations that are at least 70%-80% (w / w) pure, at least 80%-90% (w / w) pure, 90-95% pure, and at least 95%, 96%, 97%, 98%, 99%, or 100% (w / w) pure. In some embodiments, the protein is purified from cells, such as, but not limited to, CHO cells.

[0107] Administration, Compositions, and Kits Comprising Proteins As described herein, embodiments provided herein provide methods of treating Farber disease. In some embodiments, the methods include administering a therapeutically or prophylactically effective amount of one or more proteins described herein to a patient with Farber disease or a subject suspected of having Farber disease.

[0108] Treatment of a subject may include administration of a therapeutically effective amount of a protein described herein.

[0109] The protein may be provided in a kit as described herein.

[0110] The proteins can be used or administered alone or in combination with additional therapeutic agents. Examples of additional therapeutic agents include acid sphingomyelinase inhibitors (e.g., amitriptyline (Becker et al., 2010, "Acid Sphingomyelinase Inhibitors Normalize Pulmonary Ceramide and Inflammation in Cystic Fibrosis," Am. J. Respir. Cell. Mol. Biol., 42:716-724, which is incorporated herein by reference in its entirety) and ceramide synthase inhibitors (e.g., Shiffmann, S., Hartmann, D., Birod, K., Ferreiros, N., Schreiber, Y., Zivkovic, A., Geisslinger, G., Grosch, S., and Stark, H., 2012, "Inhibitors of Specific Ceramide Synthases,” Biochimie, 94(2):558-565, which is incorporated herein by reference in its entirety). The additional therapeutic agent may be a ceramidase mixture as described in U.S. Patent Application Publication No. 20160038574, which is incorporated herein by reference in its entirety.

[0111] Enzyme replacement therapy (ERT) can be effective, as shown in our current study of Farber disease, which demonstrated a reduction in ceramide accumulation, but antibodies against the drug, i.e., the replacement enzyme, can develop that can reduce its efficacy.Here, we show that repeated administration is well tolerated, which supports a treatment regimen of repeated administration of replacement enzyme, particularly the enzyme produced according to the methods described herein and, for example, in US Patent Publication No. 20160038574, that results in relief of disease symptoms.

[0112] In some embodiments, a method of treating Farber disease in a subject in need thereof comprises administering to the subject a pharmaceutical composition comprising an effective amount of recombinant human acid ceramidase about once per week, every 2 weeks, 3 weeks, or 4 weeks, or once per month, about 10 weeks, about 20 weeks, or about 30 weeks, 1 year, 5 years, 10 years, or 25 years, or once per the patient's lifetime.

[0113] Suitable vehicles and their formulation and packaging are described, for example, in Remington: The Science and Practice of Pharmacy (21st ed., Troy, D. ed., Lippincott Williams & Wilkins, Baltimore, Md. 2005) Chapters 40 and 41). Additional pharmaceutical methods may be used to control the duration of action. Controlled release formulations may be achieved by using polymers to complex or absorb the compound. Another possible way to control the duration of action by controlled release formulations is to incorporate the compound into particles of polymeric materials such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinyl acetate copolymers. Alternatively, instead of incorporating these drugs into polymer particles, these substances can be encapsulated in microcapsules prepared, for example, by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, or in colloidal drug delivery systems, such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules, or macroemulsions.

[0114] Generally, when administering a systemic dose of protein, it is desirable to provide the recipient with a dosage of protein that is in the range of about 1 ngkg-100 ng / kg, 100 ng / kg-500 ng / kg, 500 ng / kg-1 μg / kg, 1 μg / kg-100 μg / kg, 100 μg / kg-500 μg / kg, 500 μg / kg-1 mg / kg, 1 mg / kg-50 mg / kg, 50 mg / kg-100 mg / kg, 100 mg / kg-500 mg / kg (of the recipient's body weight), although lower or higher dosages may be administered.

[0115] In some embodiments, the dose of rhAC in an adult human is about 0.8 mg / kg. In some embodiments, the dose of rhAC in a child human is about 1.2 mg / kg.

[0116] In some embodiments, the effective amount of rhAC administered is about 0.1 mg / kg to about 10 mg / kg. In some embodiments, the effective amount is about 1 mg / kg to about 5 mg / kg. In some embodiments, the effective amount is about 10 mg / kg to about 50 mg / kg. In some embodiments, the effective amount is about 10 mg / kg to about 20 mg / kg. In some embodiments, the effective amount is about 20 mg / kg to about 30 mg / kg. In some embodiments, the effective amount is about 30 mg / kg to about 40 mg / kg. In some embodiments, the effective amount is about 40 mg / kg to about 50 mg / kg. In some embodiments, the effective amount is about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg.

[0117] Doses may be administered once a day, twice a day, three times a day, four times a day, once a week, twice a week, once every two weeks, or once a month. In some embodiments, doses are administered once a week. In some embodiments, doses are administered once every two weeks. Treatment may also be given on a single dose schedule, or on a multiple dose schedule, where the main course of treatment involves 1-10 separate administrations, followed by other administrations at subsequent time intervals required to maintain and / or enhance the response, e.g., once a week for 1-4 months for the second administration, with subsequent administration(s) after several months as needed. Examples of suitable treatment schedules include (i) 0, 1 month, and 6 months, (ii) 0, 7 days, and 1 month, (iii) 0 and 1 month, (iv) 0 and 6 months, or other schedules sufficient to produce the desired response expected to alleviate symptoms of the disease or reduce the severity of the disease. Other treatment schedules, such as, but not limited to, those listed above, may also be used.

[0118] In certain aspects of the disclosure, treatment is initiated at less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years of age if the subject is a newborn, or at 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-25, 1-50, 1-60, 1-70, or 1-80 years of age (e.g., 1-2 years, 1-3 years, etc.). In some embodiments, the subject is 16-61 years of age. In some embodiments, the subject is 12-69 years of age. In some embodiments, the subject is 12 years of age. In some embodiments, the subject is 19-74 years of age. In some embodiments, the subject is 19 years of age. In some embodiments, the subject is 4-62 years of age. In some embodiments, the subject is 4 years of age. In some embodiments, the subject is 7-42 years of age. In some embodiments, the subject is 7 years of age. In some embodiments, the subject is a newborn. In some embodiments, the subject is 1-6 months old. In some embodiments, the subject begins treatment at 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months. In some embodiments, the subject is 6-43 years old. In some embodiments, the subject begins treatment at age 6. In some embodiments, the subject is 5-31 years old. In some embodiments, the subject begins treatment at age 5. In some embodiments, the subject is 5-57 years old. In some embodiments, the subject is 5-29 years old. In some embodiments, the subject is 1-3 years old. In some embodiments, the subject begins treatment at age 1. In some embodiments, the subject is 10-70 years old. In some embodiments, the subject begins treatment at age 10. In some embodiments, the subject is 5-80 years old, 10-70 years old, 20-75 years old, 5-60 years old, or 5-30 years old.

[0119] In some embodiments, subjects diagnosed with Farber disease are administered about 1 mg / kg to about 5 mg / kg of rhAC or about 1 mg / kg to about 5 mg / kg of rhAC every two weeks. In one embodiment, the dosage is increased from 1 mg / kg or 2 mg / kg to 5 mg / kg at week 4. If the dosage level is not tolerated by an individual subject, the dose for that subject may be reduced from 2 mg / kg to 1 mg / kg or from 5 mg / kg to 2 mg / kg, as appropriate. The rhAC may be administered every two weeks for at least 10, 20, or 30 weeks or for the life of the subject. In one embodiment, the subject is diagnosed with Farber disease and identified as having: 1) subcutaneous nodules, and / or 2) acid ceramidase activity values ​​in leukocytes, cultured skin fibroblasts, or other biological sources (e.g., plasma) that are less than 30% of control values, and / or 3) nucleotide changes in both alleles of the acid ceramidase gene (ASAH1) that indicate possible loss of function of the acid ceramidase protein by bioinformatics, gene expression studies, and / or other methods. In some embodiments, the subject is administered rhAC every 2 weeks for 28 weeks. In some embodiments, delivery of rhAC is by intravenous infusion (e.g., saline infusion). In some embodiments, the dose is started at about 1 mg / kg and increased to about 5 mg / kg rhAC (e.g., increased to 5 mg / kg at week 4).

[0120] In some embodiments, the intravenous infusion may be 3 mL / hour over time (typically 3.5-4 hours). In some embodiments, the dose may be infused at 3% of the total volume the first hour, and the remainder of the dose may be infused over the following 2.5 hours (i.e., the second hour infusion may be approximately 40% of the nominal volume per hour until the dose is administered). No maximum time of infusion is established to allow individual tolerability to be addressed on a case-by-case basis. Infusions are administered through an in-line, low protein binding 0.2 micron filter. Doses are normalized to body weight (kg) determined at each dosing interval based on body weight collected at the previous study visit.

[0121] For example, site-specific administration may be to a body compartment or cavity, such as intra-articular, intrabronchial, intraperitoneal, intracapsular, intrachondral, intracavitary, intracavity, intracerebral, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrathoracic, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, intralesional, intravaginal, rectal, oral, buccal, sublingual, intranasal, or transdermal means.

[0122] The therapeutic compositions described herein can be prepared for parenteral (subcutaneous, intramuscular, or intravenous) or any other administration use, particularly in the form of a liquid solution or suspension. The formulation can also be suitable for an injectable formulation. In some embodiments, the injectable formulation is sterile. In some embodiments, the injectable formulation is pyrogen-free. In some embodiments, the formulation does not contain other antibodies that bind to other antigens other than those described herein.

[0123] The protein of rhAC capable of treating Farber disease or other conditions associated with rhAC activity or the use to treat a condition associated with rhA is intended to be provided to a subject in an amount sufficient to affect the reduction, elimination, or amelioration of the associated symptoms or condition. Such conditions include the symptoms of Farber disease described herein in a subject. An amount is said to be sufficient or a "therapeutically effective amount" to "affect" the reduction of symptoms if the dosage, route of administration, and administration schedule of the agent are sufficient to affect such a response. The response to the protein can be measured by analysis of the affected tissue, organ, or cells of the subject, such as by imaging techniques or ex vivo analysis of tissue samples. An agent is physiologically significant if its presence results in a detectable change in the physiology of the recipient patient. In some embodiments, an amount is therapeutically effective if it is an amount that can be used to treat, ameliorate, or suppress the symptoms of Farber disease suffered by the subject. Non-limiting examples of such amounts are provided herein, but it is not intended to be limited to such amounts if the context dictates otherwise.

[0124] In some embodiments, the efficacy of treatment is assessed by any of the following means: • Percent change from baseline in net nodule (≥5 mm) count after 28 weeks of treatment with rhAC. • Percent change from baseline in net nodule (≥10mm) count and after 28 weeks of treatment with rhAC compared with placebo. • Percent change from baseline in total nodule count (regardless of size) and after 28 weeks of treatment with rhAC compared with placebo. -Changes and percent changes from baseline in range of motion in selected joints and comparison with placebo after 28 weeks of treatment with rhAC. - Change and percent change from baseline in 6-minute walk distance and comparison with placebo after 28 weeks of treatment with rhAC. - Change and percent change from baseline in pulmonary function tests and after 28 weeks of treatment with rhAC compared to placebo. - Change and percent change from baseline in FDT scores and after 28 weeks of treatment with rhAC compared to placebo. - Change and percent change from baseline in weight-for-age and height-for-age Z-scores during 28 weeks of treatment with rhAC or placebo.

[0125] In some embodiments, the pharmacokinetics of RVT-801 after administration to Ferber mice or healthy mice at different doses is evaluated based on non-compartmental methods.Non-compartmental pharmacokinetic methods estimate the exposure to drugs by estimating the area under the curve of the concentration-time graph, in particular using the following metrics known in the art: [Table 5]

[0126] In some embodiments, the tissue-specific efficacy of the treatment is assessed by determining the tissue-specific pharmacokinetics of RVT-801 based on the non-compartmental pharmacokinetic methods described above.

[0127] In some embodiments, the human equivalent dose (HED) of RVT-801 corresponding to the effective dose in Farber mice is estimated. The non-clinical evaluation of the HED herein is based on two methods: 1) FDA guidance on scaling between non-clinical species and humans by body surface area (BSA), and 2) organ:body weight ratios between species with liver and spleen as the primary tissues of ceramide accumulation and the predominant uptake of RVT-801.

[0128] Body surface area scaling: Benchmark for the Ferber mouse MED (maximum effective dose), based on human adult or pediatric BSA and body weight, HED (where HED = animal dose * (animal body weight / human body weight) 0.33 represents a dose of approximately 0.81 mg / kg for a 60 kg adult and approximately 1.2 mg / kg for a 15 kg child.

[0129] Scaling by organ:body weight ratio: PK studies have shown that the mechanisms of clearance from the vasculature are related to uptake and / or distribution in tissues, so tissue weight to body weight ratios may affect doses that the BSA model may not predict well on its own. HED=MED*(tissue ヒト / BW ヒト ) / (organization マウス / BW マウス ), a 10 mg / kg dose in mice corresponds to approximately a 3-5 mg / kg dose in an adult human, or approximately a 4-5 mg / kg dose in a 15 kg child, based on the liver and spleen of adult and pediatric humans.

[0130] In some embodiments, the HED may be determined by combining the two scaling techniques above.

[0131] The proteins can be formulated according to known methods to prepare pharma- ceutically useful compositions, whereby these materials, or their functional derivatives, are combined in admixture with a pharma- ceutically acceptable carrier vehicle.

[0132] Also provided are kits, as described herein and below, that are useful for carrying out the embodiments described herein. In some embodiments, the kit comprises a first container that contains or is packaged in association with the polypeptide described above. The kit may also comprise another container that contains or is packaged in association with a solution that is necessary or convenient for carrying out the embodiment. The container may be made of glass, plastic, or foil, and may be a vial, bottle, pouch, tube, bag, or the like. The kit may also comprise written information, such as a procedure for carrying out the embodiment, or analytical information, such as the amount of reagent contained in the first container means. The container may be in another container device, such as a box or bag, together with the written information.

[0133] Yet another aspect provided herein is a kit for treating Farber disease. In some embodiments, the kit comprises at least one container comprising a rhAC polypeptide or a nucleic acid molecule encoding the same. In some embodiments, the kit comprises a container comprising a cell configured to express rhAC. In some embodiments, the cell is a CHO cell. In some embodiments, the kit comprises a conditioned medium from a cell expressing rhAC. In some embodiments, the conditioned medium is derived from a CHO cell.

[0134] The subject matter will now be described with reference to the following examples.These examples are provided for illustrative purposes only, and claims should in no way be interpreted as being limited to these examples, but should be interpreted as encompassing all variations that become apparent as a result of the teachings provided herein.Those skilled in the art will easily recognize various non-critical parameters that can be changed or modified to produce essentially similar results. EXAMPLES

[0135] This example provides the location of rhAC in key tissue compartments following administration across the effective dose range to enable subsequent pharmacokinetic and tissue distribution modeling, and further provides a prediction of the human equivalent dose (HED) based on the effective dose and tissue exposure, scaled by body surface area or organ weight:body weight ratio.

[0136] Example 1 The serum pharmacokinetic profile of rhAC (RVT-801) following administration of RVT-801 by intraperitoneal (ip) injection at an effective dose range was measured in healthy juvenile wild-type CD-1 mice (Asah1 P361R / P361R The parental strain of mice, (see He et al., 2017, which is incorporated by reference in its entirety) was evaluated. The generation and characterization of rhAC (RVT-801) was performed according to PCT / 2018 / 052463, filed September 24, 2018 (still unpublished), which is incorporated by reference in its entirety.

[0137] Living Protocol Male CD-1 mice approximately 3.5 weeks of age (juvenile) were administered RVT-801 as a single bolus IP injection of 1, 3, or 10 mg / kg. Blood samples for pharmacokinetic analysis were collected from three animals per time point (pre-dose, 0.5, 1, 2, 3, 4, 6 post-injection) in each dose group by cardiac puncture or other approved means to generate the maximum volume of blood sample from each mouse. Each blood sample was immediately processed in its entirety to serum for RVT-801 concentration analysis by ELISA.

[0138] RVT-801 bioanalysis by ELISA A sandwich enzyme-linked immunosorbent assay (ELISA) is suitable for the quantification of RVT-801 in mouse serum. The method uses an affinity-purified rabbit anti-recombinant human AC (rhAC, RVT-801) polyclonal antibody for capture and an affinity-purified horseradish peroxidase (HRP)-conjugated rabbit anti-rhAC polyclonal antibody for detection. RVT-801 is used to establish a standard curve and control range. The signal generated is proportional to the concentration of RVT-801 in the sample within the defined range of the assay.

[0139] The detailed 2-day assay procedure is as follows: On day 1, 96-well clear polystyrene plates were coated with 100 μL of Capture Reagent (1 μg / mL rabbit anti-rhAC in PBS) and incubated overnight at 2–8 °C. All other steps were performed on day 2. All incubations on day 2 were performed at room temperature (RT) with shaking (approximately 300–400 rpm). Briefly, plates were washed three times with 300 μL of wash buffer (1x PBS with 0.05% Tween-20, PBST) and patted with a paper towel after the final wash. Plates were then blocked with 300 μL of assay buffer (casein / TBS with 0.05% Tween20) per well for at least 1 h and washed as above. After washing, 100 μL of calibration standards, controls, and samples diluted in MRD (50x in assay buffer) were added to each well, the plate was incubated for approximately 2 h, and washed as above. Then, 100 μL of detection reagent (HRP-conjugated rabbit anti-rhAC at 0.2 μg / mL in assay buffer) was added to each well, the plate was incubated for approximately 1 h, and washed as above. After washing, 100 μL of cold substrate solution was added to each well, and the plate was incubated in the dark for up to 30 min. Color development was monitored by measuring absorbance at 650 nm (A650). The reaction was stopped based on color development when the A650 was 0.9-1.2 for the best standard, or as needed. To stop color development, 100 μL of stop solution was added to each well, and the plate was read at 450 nm with wavelength compensation set to 650 nm.

[0140] Non-compartmental pharmacokinetic analysis Noncompartmental pharmacokinetic analysis was performed with Certara WinNonlin Phoenix version 7.0. Exposure ratios and graphs were generated using Microsoft Excel and GraphPad Prism.

[0141] result To evaluate the pharmacokinetic profile of the enzyme in vivo, healthy juvenile CD-1 mice received a single injection of rhAC(RVT-801) at doses of 1, 3, and 10 mg / kg, and the RVT-801 concentrations in serum at 0.5, 1, 2, 3, 4, and 6 h after injection were determined (Figure 1). As shown in Figure 2, after a single IP administration of 1, 3, and 10 mg / kg RVT-801, serum exposure (AUC, C max ) increased in proportion to the dose.

[0142] Juvenile CD-1 mouse PK data for RVT-801 are discussed below. Two studies were conducted in juvenile CD-1 mice dosed with 10 mg / kg RVT-801: RVT-801-9013A - Serum PK characterization was focused. The following PK parameters were observed: AUC1.37 hours*μg / mL C max 1.23 hours*μg / mL T max 1.0 hours RVT-801-9013B - Focused on tissue distribution and tissue:serum exposure ratios of RVT-801. The following PK parameters were observed: AUC1.49 hours*μg / mL C max 2.17 hours*μg / mL T max 0.25 hours

[0143] Furthermore, as shown in Figure 3, the duration of RVT-801 (intraperitoneal injection, 10 mg / kg) exposure in serum of wild-type CD-1 mice correlated closely with systemic acid ceramidase (AC) activity in Farber disease mice (solid line with black circles; reproduced from International Application No. PCT / US18 / 13509, the disclosure of which is incorporated by reference in its entirety). Based on this finding, further evaluation of RVT-801 pharmacokinetics and tissue-specific distribution was performed in wild-type CD-1 mice of the same age as the juvenile Farber mice used in the efficacy studies.

[0144] Example 2 Furthermore, the pharmacokinetics and tissue-specific distribution of RVT-801 were evaluated in various organ tissues of wild-type CD-1 mice.

[0145] Tissue processing prior to analysis Tissue homogenates were prepared in CHAPS buffer by adding a protease inhibitor cocktail and processing the samples on a Qiagen TissueLyser II (3 cycles of 3 min each at a frequency of 30 Hz). Prior to ELISA detection, tissue homogenates were diluted to a standard 1 mg / mL total protein concentration. Due to the low protein content, BALF samples were processed for detection based on volume rather than protein concentration.

[0146] Quantification of RVT-801 in tissue extracts by modified ELISA An ELISA method suitable for quantification of RVT-801 mouse serum was adapted for quantification in mouse tissues (blood, liver, spleen, kidney, heart, lung, and brain).

[0147] The modified method included preparation of tissue lysates, quantification of total protein using the bicinchoninic acid (BCA) assay, and preparation of an RVT-801 standard curve in lysis buffer. The adapted method had a minimum required dilution (MRD) of 50x for mouse serum quality control samples only. Standard and research samples were not subjected to MRD. The signal generated was proportional to the concentration of RVT-801 in the sample. The lower and upper limits of quantification for the adapted assay were 0.400 ng / mL and 24.5 ng / mL, respectively. Measured RVT-801 concentrations were corrected for tissue homogenization and lysate dilution, as necessary.

[0148] Prior to performing the ELISA, feasibility studies were performed. The feasibility studies included: 1) comparison of RVT-801 standard curves prepared in RIPA buffer (150 mM sodium chloride, 50 mM Tris-HCl, pH 7.5, 0.25% (w / v) deoxycholic acid, 1% (v / v) NP-40), CHAPS buffer (150 mM sodium chloride, 50 mM Tris-HCl, pH 7.5, 2% (w / v) CHAPS), and mouse serum, 2) comparison of recovery of mouse serum control samples against RVT-801 standard curves prepared in RIPA and CHAPS buffers, 3) comparison of recovery of RVT-801 standard curves using an acid phosphatase assay kit (Sigma) after different tissue homogenization methods (incubation on ice, 3 TissueLyser cycles at 30 Hz, 5 TissueLyser cycles at 30 Hz, and sonication) using different lysis buffers (PBS, RIPA, and CHAPS). CS0740), 4) confirmation of lysosomal disruption using a β-galactosidase kit (Pierce75705) after homogenization in CHAPS buffer, and 5) analysis of recovery of tissue lysates with different concentration levels of RVT-801 against a CHAPS standard curve. All solid samples except the AM pellet were weighed in tubes prior to homogenization. An empty tube was weighed at the start of each batch and this weight was used to calculate the weight of tissue in each tube. Mouse hepatic density is known from the literature, so this value (1.051 g / mL) was used for the calculation of the lysis correction factor for liver tissue. However, all other tissues were assumed to have a density of 1 g / mL (see Figure 7). ELF concentration can be determined by applying a correction factor based on urea content to the bronchoalveolar lavage fluid (BALF) data to correct for dilution of the sample during lavage and collection.

[0149] Pharmacokinetic analysis As in Example 1, non-compartmental PK analysis was performed using Certara WinNonlin Phoenix version 7.0.

[0150] result The primary clearance mechanism of RVT-801 from the circulation is uptake into vital tissues implicated in ceramide accumulation. As shown in Figure 4, RVT-801 is distributed extensively across various organ tissues (liver, spleen, kidney, lung, and heart). RVT-801 exposure in vital tissues is significantly higher than serum and persists for an extended period of time after RVT-801 is no longer measurable in the systemic circulation. This finding may support less frequent dosing than indicated based on serum exposure. The extended duration of exposure in vital organ tissues (liver, spleen) following acute RVT-801 treatment correlates with the greatest decrease in tissue ceramide occurring beyond the period of systemic exposure.

[0151] The liver and spleen are two important organs for ceramide accumulation and RVT-801 uptake in Faber mice, as described above in Example 1. Figures 5A and 5B show the time course of RVT-801 dose response in liver and spleen tissue extracts, respectively, indicating that RVT-801 exposure in the tissues increased with dose (1, 3, and 10 mg / kg). The maximum concentration of RVT-801 was achieved in the liver at approximately 4 hours after administration and remained elevated beyond the final sampling time point (24 hours) when serum levels were undetectable. Variation between sampling time points was observed, likely due to the complexity of administration of young mice (body weight of approximately 16 g).

[0152] Furthermore, the pharmacokinetics of RVT-801 was evaluated in these two vital organs. As shown in Figure 6A, compared with RVT-801 in serum and whole blood, the AUC 0~last was increased in the liver and spleen at each effective dose. As shown in Figure 6B, the tissue:serum ratio (AUC 組織 :AUC 血清 ) demonstrated significantly higher exposure per organ relative to systemic exposure across the effective dose range. Liver:serum ratios are shown in Figure 6B.

[0153] Furthermore, the pharmacokinetics of RVT-801 in these two organs was evaluated in comparison to the pharmacokinetics in various other organs. As shown in Figure 7, RVT-801 is widely distributed in tissues associated with ceramide accumulation in Fiber mice, as described above in Example 1. Across other tissues, 10 mg / kg RVT-801 achieved tissue:serum ratios as shown in Figure 8.

[0154] Discussion: These results establish the serum and tissue exposure targets required for rhAC RVT-801 to achieve maximal efficacy endpoints in lowering tissue ceramide in Farber mice. The significantly higher and more prolonged exposure achieved in key tissues associated with ceramide accumulation (liver, spleen) supports an extended period of tissue ceramide reduction by RVT-801 above detectable levels of the enzyme in serum.

[0155] Example 3 The human equivalent therapeutically meaningful dose (HED: human equivalent dose) of RVT-801 was assessed by applying the scaling strategy discussed above using body surface area or organ tissue weight:body weight ratios.

[0156] As shown in FIG. 9, the 10 mg / kg dose of RVT-801 was scaled by body surface area (BSA) to account for general physiological trends between species based on the following formula: HED=Animal Dose x (Animal Body Weight / Human Body Weight). 0.33 (where the animal dose is 10 mg / kg). Based on a mouse dose of 10 mg / kg, the adult HED was approximately 0.8 mg / kg and the pediatric HED was approximately 1.2 mg / kg.

[0157] As shown in Figures 10 and 11, the 10 mg / kg dose of RVT-801 was scaled by tissue weight:body weight ratio to account for the relative size of each compartment of RVT-801 distribution within each species using the following formula: HED = Animal Dose x (Tissue Mass ヒト / BW ヒト ) / (tissue mass マウス / BW マウス) where the animal dose is 10 mg / kg.

[0158] As shown in Figure 12, two scaling factors based on body surface area and organ-to-body weight ratios combine to provide a conservative human equivalent dose (HED) range of approximately 1-5 mg / kg per dose. This HED range is consistent with approved doses of other commercially available enzyme replacement therapies (ERTs). (Figure 12) All dosing information for other ERTs summarized in the table in Figure 12 is from the products' respective labels.

[0159] Example 4 The objective of this study was to determine the serum and tissue pharmacokinetics of RVT-801 following single-dose treatment of juvenile male CD-1 mice with 1, 3, or 10 mg / kg administered as a bolus IP injection.

[0160] After IP administration to juvenile mice, mean serum RVT-801 concentrations were highest 0.25–1 h after dose administration for all treatment groups and then declined rapidly with an estimated half-life of 1 h. Serum RVT-801 increased with increasing dose and was max and AUC last increased more than proportionally with dose. Vz / F (apparent volume of distribution after extravascular administration) exceeded total body water, and CL / F (apparent clearance after extravascular administration) exceeded hepatic blood flow. As the dose increased from 3 to 10 mg / kg, there was a trend toward a decrease in Vz / F and CL / F, suggesting that tissue distribution and clearance were saturated.

[0161] The maximum effective dose (MED) in Ferber mice was 10 mg / kg delivered by bolus IP injection, consistent with the current study. AUC last , C max , and T max The values ​​of were determined after a single dose of 10 mg / kg in age-matched wild-type CD-1 mice and were as follows: RVT-801-9013A - Serum PK characterization focused on: AUC1.37 hours*μg / mL C max 1.23 hours*μg / mL T max 1.0 hours RVT-801-9013B - Focus on tissue distribution and tissue:serum exposure ratios of RVT-801 AUC1.49 hours*μg / mL C max 2.17 hours*μg / mL T max 0.25 hours

[0162] This study was conducted in two parts. The objective of Part A of the study was to determine the serum pharmacokinetics of RVT-801 following a single dose of 1, 3, or 10 mg / kg administered as a bolus intraperitoneal injection to juvenile male CD-1 mice. The objective of Part B of the study was to characterize the disposition of RVT-801 in key tissues involved in ceramide accumulation in the Faber mouse model following a single dose of 1, 3, or 10 mg / kg administered as a bolus intraperitoneal injection to juvenile male CD-1 mice.

[0163] RVT-801 drug substance 753-01-16-002 was used in this study. The vehicle formulation used for IP administration was sterile saline.

[0164] RVT-801 was administered by bolus intraperitoneal injection to male CD-1 mice approximately 3.5 weeks of age. A total of 108 mice were used in Part A of the study, and 102 mice were used in Part B of the study. NeoSome coordinated animal shipping and delivery dates with the supplier (Charles River Laboratories) to allow the pups to fully acclimate in the animal house prior to administration.

[0165] Dose materials were prepared by diluting the provided stock solutions of RVT-801 with sterile PBS according to Table 5.

[0166] Individual doses were calculated based on body weights recorded on the day of dose administration. Animals were dosed with RVT-801 by bolus IP injection as detailed in Table 6. [Table 6] [Table 7]

[0167] Pharmacokinetic samples were collected on the schedule detailed in Table 7, with whole blood collected from 3 animals per dose group per time point. The total volume of whole blood collected from each animal was processed to serum.

[0168] [Table 8] †Blood collection fully processed to serum.

[0169] In Part B, dosing material was prepared by diluting the provided stock solution of RVT-801 with sterile PBS according to Table 8. [Table 9]

[0170] Pharmacokinetic samples were collected on the schedule detailed in Table 9. [Table 10]

[0171] Blood collections were split to provide whole blood and serum samples (where indicated) for each animal.

[0172] Serum PK samples were stored at approximately −70°C and shipped on dry ice via overnight delivery to BioAgilytix (a bioanalytical vendor) for determination of RVT-801 concentrations by sandwich enzyme-linked immunosorbent assay (ELISA).

[0173] Livers and spleens were collected from all dose groups immediately following blood collection at the appropriate time points. Tissues were gently blotted dry and placed into pre-labeled (pre-weighed) vials. The weight of each vial containing tissue was recorded and samples were stored at -70°C until shipment. No buffers, preservatives, or antibiotics were added to the tissues and no tissue perfusion was performed at any stage of the study. For the 10 mg / kg dose group, brain, kidneys, heart, and lungs were collected and processed as described for liver and spleen. Additionally, bronchoalveolar lavage fluid (BALF) was collected from the 10 mg / kg dose group immediately following blood collection at the appropriate time points by a lung lavage technique in which 0.5 mL of phosphate buffered saline was instilled into the lungs followed by collection of approximately 0.35 mL of BALF. BALF samples were placed into suitable containers and centrifuged to generate supernatant and alveolar macrophage pellet. The supernatant was removed and placed into separate vials. Supernatants and cell pellets were stored at -70°C until shipment.

[0174] Three additional mice were used to provide drug-free matrix. Control matrix samples were used as pre-dose samples for each dose group. Sample collection, processing, and storage were performed as described above. Concentrations of RVT-801 in serum were determined using a qualified sandwich ELISA method (BioAgilytix BAL-17-333-036.01-REP). The LLOQ of the serum assay was 20 ng / mL, and the ULOQ of the assay was 1224 ng / mL.

[0175] The serum method was adapted for quantification in blood, liver, spleen, kidney, heart, lung, and brain. The modified method included preparation of tissue lysates, quantification of total protein using the bicinchoninic acid (BCA) assay, and dilution to a total protein concentration of 1 mg / mL in lysis buffer, as well as preparation of an RVT-801 standard curve in lysis buffer. Due to the low protein content, BALF samples were processed on a volumetric basis, rather than on a standardized protein concentration basis. The adapted method had a minimum required dilution factor (MRD) of 50-fold for mouse serum quality control samples only. Standard and study samples were not subjected to MRD. The signal generated was proportional to the concentration of RVT-801 in the sample. The LLOQ of the adapted assay was 0.400 ng / mL, and the ULOQ of the assay was 24.5 ng / mL. The measured RVT-801 concentrations were corrected for tissue homogenization and lysate dilution. A description of the bioanalytical method and results is provided in the final bioanalytical report.

[0176] The pharmacokinetic parameters in Table 10 were derived using non-compartmental methods using serum and tissue concentration-time composite data using Phoenix WinNonlin® version 7.0 (Certara, Princeton, NJ). The area under the concentration-time curve from time zero (pre-dose) to the last non-zero time point was calculated by a combination of linear and logarithmic trapezoidal methods. The linear trapezoidal method was used for all incremental trapezoids resulting from increasing concentrations, and the logarithmic trapezoidal method was used for those resulting from decreasing concentrations (linear up / log down method). Nominal blood sampling and tissue collection times were used in the analysis.

[0177] [Table 11]

[0178] RVT-801 concentration values ​​were received from the bioanalytical laboratory in units of ng / mL and were converted to units of μg / mL for PK analysis and reporting. Concentration data and PK parameters were reported to two decimal places. max and T last The values ​​are reported to three significant digits, except for

[0179] All concentrations below the limit of quantification (BLQ) were set to zero, and the mean concentration for each dose group at each time point was calculated and concentration-time summary statistics and mean concentration-time values ​​were reported. The mean concentration, the BLQ, was used in the noncompartmental analysis without adjustment.

[0180] Any mean concentration equal to 0 (all values ​​BLQ) was considered as BLQ and treated in the NCA as follows:

[0181] If a BLQ value occurred after a measurable concentration in a profile and was followed by a quantifiable value, it was treated as missing data.

[0182] If a BLQ value occurred at the end of the collection interval (after the last quantifiable concentration), it was treated as missing data.

[0183] C max If two consecutive BLQ values ​​occurred after the first BLQ value, the profile was considered to have ended at the first BLQ value, and any subsequent concentrations were excluded from the PK calculations.

[0184] result. Individual mouse serum and tissue concentration-time data for RVT-801 used in the Part B PK analysis are shown in Table 11. [Table 12] JPEG2025041867000018.jpg251168JPEG2025041867000019.jpg246168JPEG202 5041867000020.jpg247168JPEG2025041867000021.jpg247168JPEG20250418670 00022.jpg248168JPEG2025041867000023.jpg251168JPEG2025041867000024.j pg248168JPEG2025041867000025.jpg242168JPEG2025041867000026.jpg207168

[0185] The combined mean RVT-801 concentration-time data grouped by tissue and dose are shown in Table 12. [Table 13] JPEG2025041867000028.jpg206168JPEG2025041867000029.jpg188168

[0186] Mean RVT-801 concentration-time data for the 10 mg / kg dose group are displayed with the tissues overlaid on the linear axis in FIG.

[0187] Figures 20A and 20B show the mean RVT-801 tissue concentration-time profiles (linear and log-linear), respectively, following IP administration to young mice in part B.

[0188] After IP administration to juvenile mice, mean serum RVT-801 concentrations were highest at 0.25 hours post-dose for all dose groups. Serum RVT-801 concentrations declined rapidly. Final non-zero mean concentrations occurred at 3 hours for the 1 mg / kg dose, 4 hours post-dose for the 3 mg / kg dose, and 4 hours post-dose for the 10 mg / kg dose. Variability in serum concentrations across dose groups was large. Serum concentrations in Part B of the study were generally consistent with those in Part A.

[0189] Following a 10 mg / kg dose, RVT-801 concentrations in tissues were quantifiable at the first time point post-dose and were highest in the lungs at 0.5 hours and in the liver, spleen, kidneys, and heart at 1 hour post-dose. RVT-801 concentrations were generally quantifiable in tissues up to 18 hours post-dose.

[0190] The combined mean RVT-801 serum concentration-time data for Parts A and B grouped by dose are summarized in Tables 13 and 14, respectively. [Table 14]

[0191] [Table 15]

[0192] The composite mean non-compartmental PK parameters of RVT-801 by matrix are shown in Table 15. [Table 16]

[0193] The ratios of tissue to serum exposure are shown in Table 16 and illustrated in Figure 21. Figure 21 shows the AUC last Figure 1 shows RVT-801 tissue:serum exposure ratios based on

[0194] BALF, brain, heart, kidney, and lung were collected only for the 10 mg / kg dose group. [Table 17]

[0195] A correction factor to account for dilution is required to reflect the concentration of ELF.

[0196] Dose-normalized PK parameters plotted against dose level for various tissues are shown in Figures 22A-22D.

[0197] FIG. 22A shows dose-normalized area under the curve (DNAUC) data in blood following administration of 1 mg / kg, 3 mg / kg, and 10 mg / kg of RVT-801.

[0198] FIG. 22B shows dose-normalized area under the curve (DNAUC) data in serum following administration of 1 mg / kg, 3 mg / kg, and 10 mg / kg of RVT-801.

[0199] FIG. 22C shows dose-normalized area under the curve (DNAUC) data in liver tissue following administration of 1 mg / kg, 3 mg / kg, and 10 mg / kg of RVT-801.

[0200] FIG. 22D shows dose-normalized area under the curve (DNAUC) data in the spleen following administration of 1 mg / kg, 3 mg / kg, and 10 mg / kg of RVT-801.

[0201] Systemic serum exposure to RVT-801 was short-lived, with serum concentrations being quantifiable for a much shorter period than tissue concentrations: 4 hours in serum versus 18–24 hours in tissue. max This occurred within 1 hour of administration, consistent with rapid distribution of the compound to tissues.

[0202] RVT-801 was widely distributed in tissues, and the tissue and serum exposure (AUC last ) ranged from 0.000165 (BALF:serum) to 92.9 (liver:serum). The highest exposures were observed in liver > spleen > kidney > lung > heart > whole blood > BALF, with T max Exposures in the liver, spleen, kidneys, heart, and lungs were quantifiable for 18 to 24 hours after dosing.

[0203] BALF samples consisted of ELF (alveolar epithelial fluid) diluted with phosphate-buffered saline (PBS) infusion fluid. Cells were removed by centrifugation immediately after BALF collection, so alveolar cell populations were not present in the assay samples. A correction factor reflecting the dilution of native lung fluid in PBS is necessary to convert RVT-801 concentrations in BALF to levels in ELF. Determination of absolute dilution factors was not performed as part of this exploratory study. A correction factor can be determined by comparing the urea content in BALF with that in plasma from the same animal, since it is generally accepted that the native urea level in epithelial fluid reflects the plasma urea content (Rennard SI, Basset G, Lecossier D, O'Donnell KM, Pinkston P, Martin PG, Crystal RG, 1986, "Estimation of volume of epithelial lining fluid recovered by lavage using urea as marker of dilution," J of Applied Physiology. 1986, 60(2):532-538). Correction factors determined by Berkhout et al. using a similar collection method (Berkhout J, Melchers MJ, van Mil AC, Seyedmousavi S, Lagarde CM, Nichols WW, Mouton JW, 2015 “Pharmacokinetics and penetration of ceftazidime and avibactam into epithelial lining fluid in thigh- and lung-infected mice”,. Antimicrob Agents Chemother., 59(4):2299-2304) were highly variable (mean, 11.6-fold; range, 4.3-144.2-fold for a total injection volume of 2 mL), indicating that dilution in the present study (0.5 mL injection volume) could be up to 36-fold.

[0204] Concentrations of RVT-801 were lower in blood than in serum, with blood:serum ratios ranging from 0.127 to 0.584, suggesting that RVT-801 is not preferentially associated with red blood cells. See Table 17. [Table 18]

[0205] The objective of this study was to determine the serum and tissue pharmacokinetics of RVT-801 following single-dose treatment to juvenile male CD-1 mice of 1, 3, or 10 mg / kg administered as a bolus IP injection. Following IP administration to juvenile mice, mean serum RVT-801 concentrations were highest 0.25 to 1 hour after dose administration for all treatment groups and declined rapidly, with an estimated half-life of 1 hour. Serum RVT-801 increased with increasing dose and C max and AUC last increased more than proportionally with dose. Vz / F exceeded total body water and CL / F exceeded hepatic blood flow. As the dose increased from 3 mg / kg to 10 mg / kg, there was a trend for Vz / F and CL / F to decrease, suggesting that tissue distribution and clearance were saturated. The maximum effective dose (MED) in Faber mice was 10 mg / kg delivered by bolus IP injection, consistent with the present study. AUC determined after a single dose of 10 mg / kg in age-matched wild-type CD-1 mice was 1.2 mg / kg. last The value of C is 1.37 μg.h / mL. max was 1.23 μg / mL.

[0206] RVT-801 underwent rapid clearance from the systemic circulation, consistent with widespread distribution from serum to other tissues. The highest exposure of RVT-801 was observed in the liver > spleen > kidney > lung > heart > whole blood > BALF. AUC lastTissue to serum exposure ratios based on BALF ranged from 0.000165 (BALF:serum) to 92.9 (liver:serum). Tissue exposure generally lasted 18-24 hours after dosing. Concentrations of RVT-801 were lower in blood than in serum, with blood:serum ratios ranging from 0.127 to 0.584, suggesting that RVT-801 does not preferentially associate with red blood cells.

[0207] Example 5 Tissue distribution studies in Ferber mice aged 4-5 weeks at the time of dosing were performed utilizing a total of 12 Ferber mice (Study RVT-801-9021; Part A). Six animals each received either a single dose of 10 mg / kg RVT-801 or three weekly doses of 10 mg / kg / dose RVT-801. P361R / P361R Treatment groups consisted of animals of either sex because animal supplies were limited and no sex-specific effects of rhAC were observed in these animals. A summary of the treatment phases of the study is shown in Table 18. [Table 19]

[0208] Male and female Ferber mice, 4-5 weeks of age at the start of dosing, were given a single dose of RVT-801 or repeated weekly doses by bolus intraperitoneal (IP) injection. Dose materials were prepared as shown in Table 19. Individual doses were calculated based on body weights recorded on the day of dose administration. RVT-801 was diluted in sterile saline and dose materials were prepared fresh on each dosing day. [Table 20]

[0209] Experiments were designed to determine the concentrations of rhAC in the circulation and tissues at selected time points following single or repeated administration of RVT-801 to Faber mice, and to assess the extent and degree of RVT-801 distribution to key tissues with respect to the pharmacokinetics established in CD-1 mice.

[0210] Example 6 Immunotype studies were performed in untreated and treated Ferber mice. Untreated Ferber mice and wild type (WT) mice were dosed at 4, 6, and 8 weeks. Treated Ferber mice were dosed at approximately 4 weeks. The study utilized 11 (male / female) untreated Ferber mice, 8 (male / female) untreated WT mice, and 7 (male / female) treated Ferber mice. RVT-801 was dosed weekly. This arm of the study was designated as RVT-801-9025 Part B.

[0211] The rhAC administered in Examples 5 and 6 consisted of a 10 mg / ml (actually 9.91 mg / ml) solution of rhAC at pH 7.4.

[0212] Following dosing of RVT-801, mice were euthanized according to standard operating procedures and PK samples were collected at 6 and 24 hours post-dose (sample collection occurred after the third weekly injection of RVT-801 in the repeat-dose group). Due to limited sample availability in the Faber mice, pharmacokinetic sample collection was restricted to these two time points. Whole blood was collected by cardiac puncture into lithium heparin vials and processed whole into plasma. Intact liver, spleen, kidney, lung, heart, brain, and thymus tissues were collected from each animal, gently blotted dry, and placed into individual vials. Tissues were not fixed or perfused with buffer at any stage, and samples were frozen without the addition of buffers, preservatives, protease inhibitors, or antibiotics. Plasma and tissue samples were stored at approximately -70°C and shipped on dry ice to a bioanalytical facility for analysis. PK sample collection is detailed in Table 20. [Table 21]

[0213] Tissue-based ELISA Concentrations of rhAC in Faber mouse tissues following RVT-801 administration were measured by ELISA performed by BioAgilytix (Durham, NC).

[0214] Tissue samples were run against calibration standards prepared in homogenization buffer containing either serum samples or buffer-based quality control (QC) samples.

[0215] The resulting tissue samples were homogenized in buffer (150 mM sodium chloride, 50 mM Tris-HCl, pH 7.5, 2% (w / v) CHAPS, Thermo Scientific) containing a protease inhibitor cocktail (HALT, Thermo Scientific). Samples were processed using a TissueLyser II (Qiagen) with three cycles of 3 min each, using a single 5 mm stainless steel bead at 30 Hz. To control sample temperature, homogenates were heated to 2–8 °C between cycles. Total protein concentration of each tissue homogenate was determined with a colorimetric bicinchoninic acid (BCA) protein quantification kit (Pierce), and samples were diluted with buffer to a nominal total protein concentration of 1 mg / mL before analysis by ELISA.

[0216] The capture antibody (rabbit anti-rhAC) was coated onto a microtiter plate. The plate was blocked with a proteinaceous buffer and washed to remove excess capture and blocking reagents. Diluted tissue homogenates were added to the plate along with calibration standards and QCs and incubated to allow rhAC in the samples and controls to bind to the antibody coating the plate. The plate was washed to remove unbound rhAC and incubated with detection antibody (rabbit anti-rhAC-HRP). The plate was washed to remove unbound detection antibody and incubated with a colorimetric substrate (3,3',5,5'-tetramethylbenzidine (TMB)) to visualize rhAC bound to the detection antibody. The absorbance of the samples was read after the addition of stop solution. The assay generated a signal proportional to the concentration of rhAC in the sample using the parameters outlined in Table 21. The RVT-801 concentration in the tissue homogenates was interpolated from the standard curve and corrected by applying a dilution factor to calculate the concentration in native tissue. [Table 22]

[0217] The content of rhAC in Farber mouse plasma was determined by a qualified ELISA assay. Briefly, microtiter plates were coated with capture reagent (rabbit anti-rhAC), blocked, washed, and incubated with the resulting plasma samples and matrix-matched calibration standards and QCs. After incubation, the plates were washed to remove unbound rhAC and then incubated with detection reagent (rabbit anti-rhAC-HRP). Further washing removed excess (unbound) detection reagent, and colorimetric substrate (3,3',5,5'-tetramethylbenzidine (TMB)) was added to the plate. Finally, a stop solution was added to stop the colorimetric development, and the absorbance of the samples was read. The intensity of the colorimetric signal generated in the assay was proportional to the concentration of rhAC in the samples. See Table 22 for a summary of the assay parameters. RVT-801 concentrations in Farber mouse plasma samples were back-calculated from the standard curve. [Table 23]

[0218] The area under the RVT-801 concentration-time curve was P361R / P361R Neither half-life nor clearance was calculated in the Faber mice due to insufficient amount and time density of data points over the post-dosing period, which is a function of the limited number of animals. For similar reasons, neither half-life nor clearance was calculated.

[0219] RVT-801 concentration data are reported to three significant figures and max Values ​​were reported to two decimal places.

[0220] All resulting sample concentrations reported below the limit of quantitation (BLQ) were treated as having a value equal to zero when calculating the mean concentration-time data.

[0221] result Mean rhAC concentration-time data in plasma and selected tissues from Faber mice receiving a single dose or repeated weekly doses of 10 mg / kg / dose of RVT-801 are shown in Table 6, with individual data detailed in Table 7.

[0222] Following a single dose of RVT-801, the highest concentrations of rhAC were measured at 6 hours post-dose (the first time point post-dose) across all biological matrices collected from Faber mice, however, these data may be biased by the limited number of feasible sampling time points.

[0223] Following repeated dosing of 10 mg / kg / dose of RVT-801, rhAC levels in the circulation and across tissues collected at 6 and 24 hours post-dose were generally lower in CD-1 mice (RVT-801-9021) than those following a single dose of 10 mg / kg RVT-801, with multiple 24 hour repeat dose samples below the limit of quantification. This trend is readily apparent among the three tissues with the highest total rhAC content following RVT-801 dosing in Ferber mice (liver, spleen, and kidney), where the average single dose 24 hour concentrations were an order of magnitude greater than those following multiple doses (Figure 13). One exception to this general trend was the brain, where the only samples with quantifiable rhAC concentrations were from two mice that received multiple doses of RVT-801.

[0224] FIG. 21 shows the AUC last 1 shows RVT-801 tissue:serum exposure ratios in BALF, blood, brain, heart, kidney, liver, lung, and spleen based on 100% CI 0.01 to 0.05.

[0225] [Table 24]

[0226] Table 23: Faber (Asahel) after receiving either a single dose or repeated weekly doses of 10 mg / kg / dose of RVT-801 via bolus IP injection. P361R / P361R ) Mean rhAC concentrations across selected biological matrices in mice. Animals in RVT-801-9025 part B received four weekly doses of RVT-801, while mice receiving multiple doses in RVT-801-9021 were injected three times weekly with RVT-801. RVT-801 sample concentrations determined by sandwich ELISA. ELISA calibration ranges: 20.0-1224ng / mL (serum), 0.400-24.5ng / mL (tissue), 20.0-1280ng / mL (plasma).

[0227] BLQ: below limit of quantification NC: Not collected 1 Data from Study RVT-801-9021 2 Data from Study RVT-801-9025 Part B [Table 25]

[0228] Table 24 shows the results of Faber (Asahil) after receiving either a single dose or repeated weekly doses of 10 mg / kg / dose of RVT-801 by bolus IP injection. P361R / P361R ) shows the average rhAC concentrations across selected biological matrices in mice. Animals in RVT-801-9025 part B received four weekly doses of RVT-801, while mice receiving multiple doses in RVT-801-9021 were injected three times weekly with RVT-801. RVT-801 sample concentrations determined by sandwich ELISA. ELISA calibration ranges: 20.0-1224ng / mL (serum), 0.400-24.5ng / mL (tissue), 20.0-1280ng / mL (plasma). BLQ: below limit of quantification NC: Not collected

[0229] result. The mouse pharmacokinetics of RVT-801 was previously characterized in healthy juvenile male CD-1 mice (approximately 3.5 weeks of age) (RVT-801-9013 part A). Mice were dosed immediately after weaning to approximate the size of the Farber mice and their age at the start of efficacy studies performed in the severe Farber model. The decision to use CD-1 mice for general ADME studies was based on a common genetic background (parental strain of Farber mice) along with the cachectic nature and characteristic vulnerabilities of the severe Farber mice, which impact their suitability and availability to describe a sufficient concentration-time course of administered RVT-801.

[0230] When administered at 1, 3, or 10 mg / kg as a single IP bolus injection to healthy juvenile male CD-1 mice, systemic exposure of RVT-801 was generally max and AUC increased more than proportionally with dose, with peak serum concentrations occurring within 1 hour after dosing. Although relatively short-lived in the circulation (half-life <1 hour), the apparent volume of distribution and apparent clearance were consistent with rapid and extensive distribution of RVT-801 through the vasculature, as these values ​​exceeded total body water and hepatic blood flow, respectively (Table 25). [Table 26]

[0231] Table 25. Serum pharmacokinetics of RVT-801 following a single IP bolus injection in healthy juvenile male CD-1 mice; PK samples collected up to 24 hours post-dose, n=3 per time point. C max : Maximum observed concentration T max :Time of maximum observed concentration T last : Time of the last quantifiable time point AUC last : Area under the concentration-time curve from 0 to the last quantifiable time point t 1 / 2 Terminal half-life Vz / F: Apparent volume of distribution after extravascular administration CL / F: Apparent clearance after extravascular administration

[0232] The exposure time and general excretion kinetics of RVT-801 in CD-1 mice correlated closely with the duration and half-life of acid ceramidase activity measured in Faber mice following a single dose of rhAC at 10 mg / kg in either strain (Figure 14). This observation lent further credence to the use of CD-1 mice to characterize the murine pharmacokinetics of RVT-801 and extrapolate general ADME properties to disease models.

[0233] FIG. 15 shows the final rhAC tissue:serum AUC ratios in healthy young CD-1 mice following a single 10 mg / kg dose of RVT-801.

[0234] When administered at 1, 3, or 10 mg / kg as a single IP bolus injection to juvenile male CD-1 mice, RVT-801 was distributed extensively to a range of selected tissues and organs (RVT-801-9013 Part B). Ranking of tissues by exposure, based on AUC, shows liver > spleen > kidney > lung > heart > serum > blood (Figure 15, Table 9).

[0235] In vivo blood distribution analysis suggested that RVT-801 was not preferentially associated with red blood cells. A significantly extended period of exposure was observed in tissues compared to serum. RVT-801 was quantifiable in serum for up to 4-6 hours after dosing, whereas tissue exposure generally persisted for at least 18-24 hours after dosing.

[0236] At a dose of 10 mg / kg RVT-801 in CD-1 mice, which correlates with the maximally effective dose (MED) in the Faber mouse model based on reduced accumulated ceramide levels in the liver and spleen, the majority of quantifiable rhAC in tissues selected for analysis was distributed to the liver, spleen, and kidney. [Table 27]

[0237] Table 26. Systemic and tissue pharmacokinetics of RVT-801 following a single IP bolus injection in healthy juvenile male CD-1 mice; PK samples collected up to 24 hours post-dose, n=3 per time point. C max : Maximum observed concentration T max :Time of maximum observed concentration T last : Time of last quantifiable time point AUC last : Area under the concentration-time curve from 0 to the last quantifiable time point Comparison of the single-dose pharmacokinetics of RVT-801 in Ferber mice (limited to two time points post-dose) with the exposure profile characterized in CD-1 mice suggested high rhAC exposure in Ferber mice, with significantly increased systemic levels of rhAC and more generally high exposure across tissues at the time points analyzed following a single 10 mg / kg bolus IP injection of RVT-801 in either strain (Table 26). [Table 28]

[0238] Table 27. Healthy male CD-1 mice approximately 3.5 weeks of age and male and female Faber mice aged 4-5 weeks (Asah1 P361R / P361R ) RVT-801 pharmacokinetic parameters. A total of 33 CD-1 mice and 6 Faber mice were used (n=3 per time point for each strain); average data are reported. RVT-801 sample concentrations determined by sandwich ELISA. ELISA calibration range: 20.0-1224ng / mL (serum), 0.400-24.5ng / mL (tissue), 20.0-1280ng / mL (plasma). RVT-801 brain exposure was limited in CD-1 mice and not quantifiable in Faber mice following a single dose and is therefore not shown.

[0239] 1 Asah1 availableP361R / P361R AUC was not calculated for the Ferber mice because there were insufficient concentration-time data points over the post-dose period, which was a function of the limited number of animals.

[0240] 2 Maximum RVT-801 concentrations were measured 6 hours post-dose across all biological matrices collected from Ferber mice (previous post-dose assessment). These data may be biased by the limited number of feasible sampling time points.

[0241] 3 Due to the limited availability of Faber mice, pharmacokinetic sample collection was restricted to 6 and 24 hours post-dose.

[0242] Systemic rhAC levels measured in Farber plasma and CD-1 serum following a single 10 mg / kg IP dose of RVT-801 suggest that Farber mice experienced higher systemic rhAC exposure than CD-1 mice, as indicated by higher levels at 6 hours post-dose, which remained present in the circulation and were quantifiable up to the 24 hour concentration time point in Farber plasma, whereas rhAC was undetectable / below the limit of quantification in CD-1 mouse serum beyond 6 hours (FIG. 16).

[0243] In Farber mice, the highest concentrations of RVT-801 occurred 6 hours after administration across all biological matrices analyzed. Farber's study max Although the study was not designed to clearly define rhAC concentrations and direct comparison of rhAC concentrations at 6 hours post-dose in Faber to CD-1 mice was not done in light of the lack of CD-1 data at that particular time point, at 6 hours Faber liver, spleen, lung, and heart concentrations were significantly higher than those observed in the corresponding CD-1 tissues. maxThe exception was the kidney, where CD-1 mice showed slightly higher concentrations. At 24 h post-dose, rhAC levels in CD-1 tissues were below the limit of quantification or declined rapidly, whereas concentrations in Faber mouse tissues were significantly above the LLOQ, indicating that T in CD-1 mice was significantly higher than that in Faber mice. last remained elevated compared with serum data.

[0244] A comparison of rhAC in the circulation and in the major tissues of exposure after a single dose of RVT-801 is shown in Figure 17. A comparison of the spread of the individual data points is shown in Figure 18, where the variation in rhAC concentrations at a given time point after a single dose of RVT-801 can be appreciated across the tissues analyzed. Again, the increased exposure in Faber compared to CD-1 mice at 24 hours post-dose is readily apparent.

[0245] FIG. 17 shows concentrations of rhAC in the circulation and in the three major exposed tissues in Faber (open symbols) and CD-1 (filled symbols) mice following a single 10 mg / kg bolus IP injection of RVT-801. n=3 animals per time point. Samples were collected and analyzed up to 24 hours post-dose. Faber mouse samples were collected only at 6 and 24 hours post-dose. CD-1 spleen data at 6 hours is driven by a single quantifiable value.

[0246] Figure 18 shows individual rhAC concentration-time data in liver (Figure 18A), spleen (Figure 18B), kidney (Figure 18C), lung (Figure 18D), and heart (Figure 18E) of Faber (open symbols) and CD-1 (filled symbols) mice following a single 10 mg / kg bolus IP injection of RVT-801. n=3 per time point. Samples were collected and analyzed up to 24 hours post-dose.

[0247] Young, healthy CD-1 mice are considered the parental strain for the Farber disease mouse model. Figure 3 shows that the RVT-801 pharmacokinetic profile in young CD-1 mice is similar to the experimental rhAC activity profile in the blood of Farber mice, indicating that young CD-1 mice adequately approximate the PK of Farber mice (because Farber mice are fragile, difficult to breed, and not available in sufficient numbers to perform PK evaluation).

[0248] Although based on a small number of Farber mice and with limited overlapping data points between strains, the data suggest that systemic rhAC exposure was significantly higher in Farber compared to CD-1 mice following a single 10 mg / kg dose of RVT-801. The resulting systemic PK samples differed in a number of potentially important ways. Farber mice were mixed-sex, significantly smaller than healthy, age-matched CD-1 mice, exhibited severe pathology, and generated plasma samples, whereas serum was collected from juvenile male CD-1 mice.

[0249] The observed differences in RVT-801 exposure are likely not a result of analyzing different matrices between the strains, as the protein content (with the exception of clotting factors) of serum and plasma is nearly identical. Furthermore, the increased Farber plasma exposure measured was not a result of increased plasma ELISA sensitivity compared to ELISA sensitivity in serum, and / or the ability to report additional data points (i.e., 24 hours post-dose) that were undetectable in CD-1 serum due to differences in method sensitivity. The Farber plasma data reported were well above the LLOQ, and the plasma and serum methods performed similarly (Table 27).

[0250] [Table 29]

[0251] The tissue RhAC concentration after treatment did not appear to depend on the tissue weight to body weight ratio in Ferber mice. a .

[0252] a Based on repeated dose Faber animals for RVT-801-9025; sex and weight were not recorded for RVT-801-9021.

[0253] Significant immunological differences between wild-type and Ferber mice have been demonstrated, but their impact on the pharmacokinetics of RVT-801 is unclear. Anti-drug antibodies (ADAs) can prolong the circulation of drug-antibody complexes, but the PK differences under investigation were observed within 24 hours after administration of a single dose of RVT-801, and no ADAs were detected in these samples.

[0254] The difference in exposure observed in Farber mice compared with CD-1 mice may have been a result of multi-organ physiological changes in the Farber disease mouse model.

[0255] Differences in the percentage distribution of RVT-801 from the peritoneal cavity to the vasculature and / or differences in uptake of rhAC from the circulation to tissues between Faber and CD-1 mice could explain the PK differences observed between strains. However, these potential trends cannot be assessed from the present study without significant overinterpretation of the limited available data.

[0256] There was no obvious accumulation of rhAC upon repeated weekly dosing of RVT-801 compared to the RVT-801-9021 dose group. Rather, the data, although limited, suggest that multiple weekly doses of RVT-801 resulted in lower rhAC concentrations in the circulation and across the tissues analyzed compared to a single injection at the same dose level (10 mg / kg / dose). Across all matrices except brain, single dose concentration-time data at 6 and 24 hours post-dose were higher after a single dose of RVT-801 than after three weekly doses (Figures 19A-G). Furthermore, the data are consistent with a scenario of lower exposure due to both lower absolute rhAC concentrations and higher clearance rates after multiple doses of RVT-801. Further studies will be required to investigate these apparent trends and establish whether the data are consistent with general mouse pharmacokinetics of RVT-801 upon repeated dosing or whether pathology features specific to the Farber disease mouse model affect the uptake, disposition, and clearance of rhAC.

[0257] Figures 19A-G show the mean rhAC concentrations in CD-1 mice after a single dose of 10 mg / kg by bolus IP injection and in Faber mice after either a single dose of 10 mg / kg or multiple weekly doses of 10 mg / kg / dose according to Example 5 (RVT-801-9021).

[0258] Figures 20A and 20B show the mean rhAC tissue concentration-time profiles following IP administration to young CD-1 mice in RVT-801-9013 part B, respectively (linear Figure 20A and log-linear Figure 20B).

[0259] FIG. 21 shows the AUC last Figure 1 shows RVT-801 tissue:serum exposure ratios in BALF, blood, brain, heart, kidney, liver, lung and spleen based on 100% CI 0.01 to 0.05.

[0260] Figures 22A-D show dose-normalized PK parameters plotted against dose level for various tissues following IP administration of RVT-801 at doses of 1 mg / kg, 3 mg / kg, and 10 mg / kg in CD-1 mice.

[0261] FIG. 19A shows a plot of systemic rhAC concentrations in serum after a single dose of 10 mg / kg / dose by bolus IP injection of RVT-801. FIG. 19A also shows plasma concentrations in Faber mice after a dose of 10 mg / kg / dose by bolus IP injection of RVT-801. Additionally, FIG. 19A shows plasma concentrations after multiple doses by injection of RVT-80. Also shown is serum concentration in CD-1 mice administered a single dose of 10 mg / kg / dose by bolus IP injection of RVT-801.

[0262] Figure 19B shows the concentration of RVT-801 in liver tissue of CD-1 mice after a single dose of 10 mg / kg / dose by bolus IP injection of RVT-801. Figure 19B also shows the concentration of RVT-801 in liver tissue of Ferber mice treated with a single dose of 10 mg / kg / dose by bolus IP injection of RVT-801. Figure 19B further shows the concentration of RVT-801 in liver tissue of Ferber mice treated with repeated doses of 10 mg / kg / dose by bolus IP injection of RVT-801.

[0263] Figure 19C shows the concentration of RVT-801 in spleen tissue of CD-1 mice after a single 10 mg / kg / dose by bolus IP injection of RVT-801. Figure 19C also shows the concentration of RVT-801 in spleen tissue of Faber mice treated with a single 10 mg / kg / dose by bolus IP injection of RVT-801. Figure 19C further shows the concentration of RVT-801 in liver tissue of Faber mice treated with repeated doses of 10 mg / kg / dose by bolus IP injection of RVT-801.

[0264] Figure 19D depicts the concentration of RVT-801 in kidney tissue of CD-1 mice after a single dose of 10 mg / kg / dose by bolus IP injection of RVT-801. Figure 19D also depicts the concentration of RVT-801 in kidney tissue of Ferber mice treated with a single dose of 10 mg / kg / dose by bolus IP injection of RVT-801. Figure 19C further depicts the concentration of RVT-801 in kidney tissue of Ferber mice treated with repeated doses of 10 mg / kg / dose by bolus IP injection of RVT-801.

[0265] Figure 19E shows the concentration of RVT-801 in the cardiac tissue of CD-1 mice after a single 10 mg / kg / dose by bolus IP injection of RVT-801. Figure 19E also shows the concentration of RVT-801 in the cardiac tissue of Ferber mice treated with a single 10 mg / kg / dose by bolus IP injection of RVT-801. Figure 19E further shows the concentration of RVT-801 in the cardiac tissue of Ferber mice treated with repeated doses of 10 mg / kg / dose by bolus IP injection of RVT-801.

[0266] Figure 19F depicts the concentration of RVT-801 in lung tissue of CD-1 mice after a single 10 mg / kg / dose by bolus IP injection of RVT-801. Figure 19F also depicts the concentration of RVT-801 in lung tissue of Ferber mice treated with a single 10 mg / kg / dose by bolus IP injection of RVT-801. Figure 19F further depicts the concentration of RVT-801 in lung tissue of Ferber mice treated with repeated doses of 10 mg / kg / dose by bolus IP injection of RVT-801.

[0267] Figure 19G shows the concentration of RVT-801 in brain tissue of CD-1 mice after a single dose of 10 mg / kg / dose by bolus IP injection of RVT-801. Figure 19G further shows the concentration of RVT-801 in brain tissue of Faber mice treated with repeated doses of 10 mg / kg / dose by bolus IP injection of RVT-801.

[0268] Abbreviation. ASAH1 = acid ceramidase gene.

[0269] AUC=area under the concentration-time curve.

[0270] AUC (0-∞) = area under the plasma concentration-time curve extrapolated from time zero to infinity.

[0271] AUC (0~t) = area under the plasma concentration-time curve from time zero to the last sampling time.

[0272] BMI=Body mass index.

[0273] BW=body weight.

[0274] cDNA = complementary deoxyribonucleic acid.

[0275] CFR = Code of Federal Regulations.

[0276] CI=confidence interval.

[0277] CL=clearance.

[0278] Cmax=maximum concentration.

[0279] CNS = central nervous system.

[0280] FDA=Food and Drug Administration.

[0281] HED = human equivalent dose.

[0282] HSCT=hematopoietic stem cell transplant.

[0283] IP=intraperitoneal.

[0284] IV=intravenous.

[0285] MCP-1 = monocyte chemotactic protein 1.

[0286] PD=pharmacodynamic(s).

[0287] PK=pharmacokinetic(s).

[0288] PRN=if required.

[0289] rhAC = recombinant human acid ceramidase.

[0290] SD=standard deviation.

[0291] SMA-PME=spinal muscular atrophy with progressive myoclonic epilepsy.

[0292] t max = time to maximum concentration.

[0293] References discussed in this application are incorporated by reference in their entirety for any intended purpose that is clear based on the context.

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[0348] The disclosures of all patents, patent applications, publications, and accession numbers cited herein are hereby incorporated by reference in their entireties.

[0349] While the present disclosure has been disclosed with reference to various embodiments, it will be apparent that other embodiments and variations thereof may be devised by those skilled in the art without departing from the true spirit and scope of the present disclosure, and it is intended that the appended claims be construed to include all such embodiments and equivalent variations.

[0350] equivalent The foregoing specification is believed to be sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and examples detail certain embodiments and explain the best mode contemplated by the inventors. However, no matter how detailed the foregoing appears in text, it is understood that the embodiments can be practiced in many ways and should be interpreted according to the appended claims and their equivalents.

[0351] The term about, as used herein, refers to numerical values, including, for example, integers, fractions, and percentages, whether or not explicitly stated. The term about refers to a range of numerical values ​​(e.g., + / - 5 to 10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). When terms such as at least and about are used before a recitation of numerical values ​​or ranges, the term modifies all values ​​or ranges provided in the recitation. In some cases, the term about may include numerical values ​​that are rounded to the nearest significant figure.

Claims

[Claim 1] 1. A method of treating Farber disease in a human subject in need thereof, comprising administering to the human subject an activated form of purified recombinant human acid ceramidase (rhAC) at a dose of about 1 mg / kg to about 10 mg / kg.

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