TW202402291A - Methods of administering belumosudil for treatment of chronic graft versus host disease in patient subpopulations - Google Patents

Abstract

The present disclosure provides methods of administering belumosudil mesylate salt to certain subpopulations of patients with cGVHD.

Description

Methods of Administering Berusudil to Treat Chronic Graft-versus-Host Disease in a Subpopulation of Patients

This disclosure relates to the administration of berusuudil mesylate (REZUROCK™) to treat chronic graft-versus-host disease (cGVHD) in certain patient subpopulations.

Chronic graft-versus-host disease (cGVHD) is an immune-mediated inflammatory and fibrotic disorder. It is a potentially serious complication after solid organ transplantation and allogeneic hematopoietic cell transplantation (alloHCT). cGVHD affects up to 70% of all alloHCT recipients, with an incidence of 20%-50% in children. It is the leading cause of non-recurrent death more than 2 years after alloHCT. The estimated prevalence of cGVHD in the United States is 14,000 patients (as of 2016). (Bachier CR et al.: Epidemiology and real-world treatment of chronic graft-versus-host disease post allogeneic hematopoietic cell transplantation: A US claims analysis. Held in Orlando, FL, December 7-10, 2019 Published at ASH 2019) ("Bachier et al.").

The quality of life (QOL) of patients with cGVHD is severely impaired, as assessed by the Lee Symptom Scale (LSS), which measures the impact of cGVHD on a patient's functioning and health. It has been reported that only one-third of patients with cGVHD who initiate systemic therapy will be alive, in remission, and discontinue immunosuppressive therapy by 5 years. (Lee SJ et al.: Success of immunosuppressive treatments in patients with chronic graft-versus-host disease . Biol Blood Marrow Transpl 24:555-562, 2018) ("Lee et al.").

The pathophysiology of cGVHD can be divided into three stages: early inflammation due to tissue damage, dysregulation of the adaptive immune system, and chronic inflammation and abnormal tissue repair with fibrosis.

First-line therapy for moderate-to-severe chronic graft-versus-host disease (cGVHD), as defined by the National Institutes of Health (NIH), is corticosteroids alone or in combination with sirolimus or a calcineurin inhibitor. However, up to 70% of patients require additional lines of therapy. (Bachier CR et al.). Furthermore, long-term use of corticosteroids is associated with significant side effects. (Lee et al.).

The management of cGVHD continues to evolve with the emergence of targeted therapies. cGVHD is characterized by overproduction of the proinflammatory cytokines IL-21 and IL-17 and overactivation of T follicular helper cells and B cells, which in turn leads to overproduction of antibodies.

In 2017, the U.S. Food and Drug Administration approved ibrutinib, a Bruton's tyrosine kinase inhibitor, for the treatment of adults with cGVHD after failure of one or more lines of systemic therapy. One study using ibrutinib in patients with cGVHD requiring an erythematous rash >25% body surface area or an NIH oral score >4 reported an overall response rate (ORR) of 67% and was limited by treatment-emergent adverse events ( TEAE)-induced discontinuation rate was 43%. (Waller EK et al.: Ibrutinib for chronic graft-versus-host disease after failure of prior therapy: 1-Year update of a phase 1b/2 study . Biol Blood Marrow Transpl 25:2002-2007, 2019).

There remain opportunities to investigate other treatment options for patients with cGVHD, including female patients of reproductive potential and male patients of reproductive potential who have female partners.

The present disclosure provides methods of administering berusudil (in some embodiments, berusudil mesylate (REZUROCK™)) to certain at-risk patient subpopulations for the treatment of cGVHD.

In one embodiment, the present disclosure provides 2-{3-[4-(1H-indazol-5-ylamine)-2-quinazolinyl]phenoxy}-N-(propan-2- The use of acetamide, or a pharmaceutically acceptable salt thereof, for the treatment of cGVHD in male or female patients of reproductive potential, including advising said patients during treatment and after the last dose of the compound Use effective contraception for at least one week.

In another embodiment, the present disclosure provides 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2 Acetamide or a pharmaceutically acceptable salt (compound) thereof (in some embodiments, its mesylate (berusudil)) in the treatment of female patients of reproductive potential with cGVHD The use includes the step of verifying the pregnancy status of said patient before initiating treatment with a compound.

In another embodiment, the present disclosure provides the use of berusudil in the treatment of chronic graft-versus-host disease (cGVHD) in a lactating patient, comprising advising the patient during treatment with the compound and at the last Do not breastfeed for at least one week after dosing the compound.

In other embodiments, the present disclosure provides use of treating chronic graft-versus-host disease (cGVHD) in a patient at risk for pregnancy, comprising the step of informing said patient of the reproductive risks associated with treatment with a compound.

In another embodiment, the present disclosure relates to a method of treating a patient suffering from cGVHD, comprising the step of verifying whether the patient is a reproductive risk patient prior to treatment. If the patient is verified not to be a reproductive risk patient, the present disclosure further provides for administering a compound to the patient; or alternatively, if the patient is verified to be a reproductive risk patient, informing the patient to receive Potential reproductive risks of treatment with berusudil; and/or Use or recommend the use of effective contraception during treatment with berusudil and for at least one week after receiving its last dose.

Embodiments of the invention may be understood more fully by reference to the detailed description and examples, which are intended to illustrate, non-limiting embodiments.

Overview

Begrusudil is an oral, selective Rho-related coiled-coil-containing protein kinase 2 (ROCK2) inhibitor. ROCK2 inhibition acts on a dysregulated adaptive immune system and fibrosis that occurs due to abnormal tissue repair. Begrusudil inhibits ROCK2 and ROCK1 with _IC50 values of approximately 100 nM and 3 µM, respectively.

Begrusudil downregulates pro-inflammatory responses via modulating STAT3/STAT5 phosphorylation and altering Th17/Treg balance in ex vivo or in vitro human T cell assays. In vitro, berusudil also inhibits aberrant profibrotic signaling. By controlling ROCK2 activity, berusudil mediates immune cell function and signaling in fibrotic pathways, thereby mitigating the effects of this debilitating disease, such as inflammation in multiple tissues and potentially involving the lungs, hepatobiliary system, Fibrotic changes in multiple organs including the musculoskeletal system, gastrointestinal (GI) tract, and skin.

Berusudil mesylate is marketed in the United States and other countries as REZUROCK ^TM for the treatment of patients with chronic GVHD (cGVHD) who, in some cases, have failed at least two prior lines of systemic therapy patients with chronic GVHD. The chemical name of the compound berusudil is as follows: 2-{3-[4-(1 H -indazol-5-ylamine)-2-quinazolinyl]phenoxy}- N -(propan- 2-yl)acetamide. The compound berusudil is also known as KD025. The active pharmaceutical ingredient of REZUROCK ^TM is berusudil mesylate, whose molecular formula is C ₂₇ H ₂₈ N ₆ O ₅ S, molecular weight is 548.62 g/mol, and chemical name is 2-{3-[4-( 1H -indazol-5-ylamine)-2-quinazolinyl]phenoxy} -N- (propan-2-yl)acetamide methanesulfonate (1:1).

The chemical structure of berusudil mesylate is as follows:

Berusuudil and processes used to prepare the compounds are described in the following US patents: US Patent No. 8,357,693, US Patent No. 9,815,820, US Patent No. 10,183,931, and US Patent No. 10,696,660.

Applicants herein have evaluated berusudil in multiple rat and rabbit embryo-fetal toxicology studies as described in Examples 1-5 below (i.e., Example 1, entitled "In Sprague-Dawley" - Preliminary Prenatal Developmental Toxicity and Toxicokinetic Studies in Dürer Rats"; Example 2, titled "Preliminary Prenatal Developmental Toxicity and Toxicokinetics in New Zealand White Rabbits with a Non-Pregnant Dose Range Discovery Phase Study"; Example 3, titled "Embryo-fetal developmental toxicity study with toxicokinetic evaluation in Sprague-Dürer rats"; Example 4, titled "Study in New Zealand White Rabbits with "Embryo-Fetal Developmental Toxicity Study for Toxicokinetic Evaluation"; and Example 5, entitled "Combined Study of Fertility and Early Embryonic Development to Implantation in Sprague-Dürer Rats").

Based on the findings from the above studies, berusudil has the potential to cause harm to the fetus when administered to pregnant women. In the above animal studies, oral doses of 25, 50, 150 and 300 mg/kg/day were administered during the organogenesis phase in the pilot study (Example 1 herein) and at oral doses of 25, 50, 150 and 300 mg/kg/day in the pivotal study (Example 3 herein). Berusudil was administered to pregnant rats at doses of 15, 50, and 150 mg/kg/day. In pilot studies, maternal toxicity and embryo-fetal developmental effects were observed. Maternal toxicity (decreased weight gain) occurred at doses of 150 and 300 mg/kg/day. Increased post-implantation losses occurred at 50 and 300 mg/kg/day. Fetal malformations including absence of anus and tail, omphalocele, and dome-shaped head were observed at ≥ 50 mg/kg/day. Rat exposure (AUC) at 50 mg/kg/day was approximately 3 times greater than human exposure at the recommended human dose of 200 mg.

In an embryo-fetal development study in rabbits (Example 4 herein), oral doses of berusudil administered to pregnant animals during organogenesis at 50, 125, and 225 mg/kg/day resulted in maternal toxicity and Effects on embryonic-fetal development. Maternal toxicity (weight loss and death) was observed at doses ≥ 125 mg/kg/day. Embryo-fetal effects, including spontaneous abortion, increased post-implantation loss, decreased percentage of viable fetuses, malformations, and reduced fetal weight, were observed at doses ≥ 50 mg/kg/day. Deformities include those of the tail (shortness), ribs (branching, fusion, or deformation), sternum (fusion), and neural arches (fusion, malpositioning, and deformation). The rabbit exposure (AUC) at 50 mg/kg/day was approximately 0.07 times the human exposure at the recommended dose of 200 mg.

In a combined male and female rat fertility study (Example 5 herein), berusudil-treated male animals were mated with untreated female animals, or untreated male animals were mated with Mating of berusudil-treated females. Berusudil was administered orally to male rats at doses of 50, 150, or 275 mg/kg/day 70 days before and throughout the mating period, and 14 days before and until gestation day 7 Orally administered to female rats. Adverse findings in female rats (treated with begrusudil or untreated but mated with treated males) at the 275 mg/kg/day dose included increased pre- or post-implantation loss and viable embryos The quantity decreases. Administration of berusudil to male rats at a dose of 275 mg/kg/day resulted in sperm abnormalities (decreased motility, decreased counts, and increased percentage of abnormal sperm) and testicular/epididymal organ changes (weight loss and degeneration) . At a dose of 275 mg/kg/day, fertility was reduced in either treated males or females and reached statistical significance in males. Adverse changes in male and female reproductive organs also occurred in general toxicology studies; findings included sperm degeneration in dogs at a berusudil dose of 35 mg/kg/day and in rats at 275 mg/kg/day. Follicle development in the ovaries was reduced at doses of Changes are partially or completely reversed during recovery. The exposure (AUC) in dogs at a dose of 35 mg/kg/day and in rats at a dose of 275 mg/kg/day was 0.5 and 8-9 times the clinical exposure at the recommended dose of 200 mg per day, respectively.

The present disclosure provides for the administration of berusuudil (in some embodiments, berusuudil mesylate (REZUROCK™)) to certain patients, including women of reproductive potential and partners at risk for pregnancy subpopulation method. This subgroup also includes nursing women due to the potential for adverse reactions in children who are breastfed by patients taking berusudil. definition

"About" as used herein includes the exact amounts modified by the term about as well as amounts expected to be within experimental error, such as within 15%, 10%, or 5%. For example, "about 200 mg" means "200 mg" and a range of mg within experimental error (eg, plus or minus 15%, 10%, or 5% of 200 mg). As used herein, the term "about" may be used to modify a range as well as a specific value.

"Administer" or "administer to" as used herein (e.g., with respect to the administration of an API (including a compound or berusudil) to a subject) means prescribing one or more drugs containing the API to the subject The act of taking by a subject during treatment, the act of dispensing said one or more drugs to said subject, and/or the act of physically receiving or ingesting said one or more drugs. Thus, an API (e.g., a compound or berusudil) may be "administered" by a physician or other medical professional who writes a prescription for one or more drugs; and/or by filling said prescription and/or administering it to a subject "Administered" by a pharmacist who dispenses said drug(s); and/or "administered" by a patient or subject who ingests said drug(s) and/or his or her partner or caregiver.

"API" means "active pharmaceutical ingredient".

"Allogeneic hematopoietic stem cell transplantation (allo-HSCT)" (also called bone marrow transplant or stem cell transplant) or "allogeneic hematopoietic cell transplantation (allo-HCT)" refers to the transplantation of hematopoietic cells from a donor into a non-identical twin. program in the receptor. The source of hematopoietic stem cells for allogeneic transplantation can be peripheral blood stem cells (PBSC) or bone marrow (BM). In some cases, umbilical cord blood may be used. The donor and recipient may have human leukocyte antigen (HLA) genetic matches, such as siblings. The donor and recipient may be only half-matched (haploidentical) parent and child.

Unless the context clearly indicates otherwise, "berusudil" as used refers to the compound berusudil in any form as well as pharmaceutically acceptable salts thereof. The term "berusudil" refers to both the compound berusudil (e.g., the free base form, the amorphous form, or the crystalline form) and a pharmaceutically acceptable salt of berusudil (e.g., as in REZUROCK mesylate form as used in ^TM ), also refers to any form of berusudil that can be used in a formulation or pharmaceutical composition for administering the compound to a patient.

"Breastfeeding" means the delivery of milk produced by lactation to a child and includes administering milk from the breast to the child and/or collecting breast milk and delivering it by other means (eg, from a bottle or container).

A "clinical endpoint" or "study endpoint" refers to an event or outcome in a clinical trial that can be objectively measured to determine the outcome and potential beneficial effects of a drug or regimen of administration as designed in the clinical trial. Examples of clinical endpoints include the following. Overall response rate (ORR) is the percentage of people in a study or treatment group who have a partial response (PR) or complete response (CR) to treatment over a certain period of time. Failure-free survival (FFS) is defined as the time from the first dose of berusudil to the failure event, or the initiation of berusudil and the addition of a new cGVHD therapy, recurrence of underlying disease, or non-relapse death (NRM) interval. Overall survival (OS) refers to the length of time from the date of diagnosis of disease or initiation of treatment. Duration of response (DOR) means the time from initial response (eg, PR or CR) until documented progression of optimal response from cGVHD, the time from initial response to initiation of additional systemic cGVHD therapy, or death. Time to next treatment (TTNT) means the time to start subsequent systemic cGVHD therapy.

"Clinically recommended amount" or "clinically recommended dose" refers to the amount or dose of an API that one skilled in the art of medicinal chemistry recommends and/or approves for administration to patients following clinical trials to treat the disease state in question. In some embodiments, the clinically recommended dose of berusudil is 200 mg once daily with food until progression of chronic GVHD requires new systemic therapy.

As used in the claims and examples herein and when clear from the context of use, " compound " is synonymous with the above all-inclusive definition of berusudil.

"CYP3A" refers to the CYP3A family of p-450 isoenzymes, including CYP3A4.

"Effective contraception" is the use of artificial methods or other techniques that effectively prevent pregnancy resulting from sexual intercourse. Examples of contraception include barrier methods, such as condoms; hormonal methods (e.g., birth control pills); intrauterine devices, such as the IUD; and male or female sterilization. The definition of effective contraception takes into account the simultaneous use of multiple forms of contraception and the increased effectiveness of contraception.

"Fetus" as used herein encompasses an unborn offspring during development, including a fertilized egg, embryo, or fetus at a later stage of development.

"Pregnancy" means the period of intrauterine development from conception to birth

"Immunosuppressive therapy" (IST) refers to therapy usually given for at least six months after allo-HSCT to try to prevent GVHD. Examples of ISTs include sirolimus, prednisone, and calcineurin inhibitors (such as tacrolimus and cyclosporine).

"Lactating patient" means a patient capable of producing breast milk.

The Lee Symptom Scale (LSS) total score measures the impact on patient functioning and health. The Lee Symptom Scale is a 30-item scale developed to measure symptoms of cGVHD and is described in Lee SJ et al., Development and validation of a scale to measure symptoms of chronic graft-versus host disease . Biol Blood Marrow Transplant 2002; 8:444-452.

A "line of treatment" or "line of therapy" describes the order or sequence in which different therapies are given to a patient as the patient's disease progresses. Initial treatment (first-line therapy) may not work or may stop working after a while. After discontinuation of first-line therapy, a second, different treatment may be given (second-line therapy). Subsequent lines of therapy may be given when second-line therapy does not work or stops working. Some patients may be given multiple lines of therapy over the course of the disease.

First-line therapy for moderate-to-severe chronic graft-versus-host disease (cGVHD), as defined by the National Institutes of Health (NIH), can be corticosteroids alone or in combination with sirolimus or a calcineurin inhibitor. (Carpenter PA et al.: A phase II/III randomized, multicenter trial of prednisone/sirolimus versus prednisone/sirolimus/calcineurin inhibitor for the treatment of chronic graft-versus-host disease : BMT CTN 0801. Haematologica 103:1915-1924, 2018 ).

Examples of corticosteroid therapies used to treat cGVHD include, but are not limited to, prednisone, prednisolone, methylprednisolone, and budesonide. Examples of prior systemic therapies used to treat cGVHD include, but are not limited to, prednisone, tacrolimus, extracorporeal photopheresis (ECP), sirolimus, ibrutinib, ruxolitinib, mycophenolate mofetil ester (MMF), rituximab, methotrexate (MTX), cyclosporine, imatinib, ixazomib, and ofatumumab.

A "deformity" is a permanent deviation that is usually incompatible with or seriously detrimental to normal postnatal survival or development. "Deformity" means a structural abnormality that alters general body conformity, disrupts or interferes with body function, or is generally considered incompatible with life. Specific examples of deformity-causing processes include marked or gross malformation, asymmetry, or abnormality of structures resulting from fusion, fission, disarticulation, malalignment, holes, enlargement, elongation, thickening, thinning, or branching. rules. Missing parts or entire structures are also considered deformities.

"Myeloablative transplantation" refers to a transplantation procedure that uses very high doses of chemotherapy or radiation before transplanting autologous or allogeneic hematopoietic stem cells. Nonmyeloablative transplantation, or reduced-intensity transplantation, involves patients receiving less intensive chemotherapy before transplantation of allogeneic hematopoietic stem cells.

The "NIH Pulmonary Symptom Score" or "NIH cGVHD Pulmonary Score" is a score based on clinical symptoms that ranges from 0 to 3. A score of 0 is for no symptoms, a score of 1 is for symptoms of shortness of breath when climbing stairs, and a score of 2 Use for symptoms of shortness of breath when on level ground, and 3 points for symptoms of shortness of breath or need for oxygen when resting.

Unless the context otherwise requires, "or" is used inclusively (equivalent to "and/or").

"Patient" as used herein includes an animal or a human, and, in one embodiment, includes a human in need of treatment with or in need of treatment with begrusudil or as a result of treatment with begrusudil or Persons who are candidates for treatment with begrusudil.

"Pregnancy" or "pregnancy" as used herein means the state of a female patient having a fertilized egg, embryo, or fetus developing in her uterus, and encompasses all stages of fetal development from the time of conception until birth.

As used herein, a "pregnancy risk patient" means any patient whose age, gender, and/or living circumstances pose a risk to his or her own pregnancy or a risk to another person's pregnancy. Therefore, "patients at risk of pregnancy" include female patients of reproductive potential, as well as male patients who are sexually active with one or more females of reproductive potential. The term "pregnancy risk patient" does not include pregnant patients.

"Pregnancy risk partner" as used herein means any person who is in a relationship with a pregnancy risk patient who may create a risk of pregnancy in the patient by any means (eg, via natural means or in vitro fertilization).

"Reproductive potential" when used with respect to a female patient means that the patient is physiologically capable of producing eggs (eggs or oocytes) and/or capable of carrying a fetus in the womb (e.g., having been implanted via artificial insemination). Female patients of “reproductive potential” do not include women who are confirmed or verified (e.g., by laboratory testing) to be pregnant. "Reproductive potential" when used with respect to a male patient means a male who is able to produce sperm to fertilize a female egg cell.

"Reproductive risks" include the risk of adverse effects on fetal or embryonic development, including, for example, weight loss, injury and/or malformation, as well as reproductive risks and risks to children breastfed from the patient under treatment.

"Reproductive risk patient" means a patient who is capable of causing or producing a reproductive risk as defined herein, including a female patient of reproductive potential, a male patient of reproductive potential, a lactating patient, or a pregnant patient.

"Risk" as used herein, for example in the definitions of "pregnancy risk patient" and "pregnancy risk partner" means that there is a possibility (even if slight or remote) that pregnancy will occur.

"Side effect" means a physiological response other than the desired effect attributable to treatment. In certain embodiments, side effects may include embryo-fetal developmental effects and malformations. Side effects can be detected directly or indirectly.

"Steroid-refractory" (SR) cGVHD is defined as cGVHD that progresses while on steroids or corticosteroids (in one embodiment, on prednisone).

"Subject" means an animal being treated with begrusudil, including an animal or human subject.

A "therapeutically effective amount" of an API means an amount that, when administered to a human to treat a disease (eg, cGVHD), is sufficient to effect treatment of the disease state being treated. As applied to cGVHD in humans, "treating" or "treatment" includes (1) reducing the risk of developing cGVHD and/or inhibiting cGVHD, that is, preventing or reducing the development of cGVHD or its clinical symptoms; (2) Relieve cGVHD, that is, cause the regression, reversal or improvement of cGVHD or reduce the number, frequency, duration or severity of its clinical symptoms.

The therapeutically effective amount of an API may vary depending on the health and physical condition of the subject to be treated, the extent of disease progression, assessment of the medical condition and other relevant factors. It is expected that a therapeutically effective amount may fall within a range that can be determined experimentally and by reference to clinical trial data and results, for example, as described in Examples 1 and 2 herein and in the scientific literature.

"Verification" as used herein with respect to the steps of verifying the pregnancy status of a female subject includes any form or manner of investigation to determine the pregnancy status of a female subject, including through physical examination, questioning, and/or one or more diagnostic tests. It is understood that a person may confirm a female subject's pregnancy by having an interview, having a physical examination, asking about a diagnostic test being performed, ordering or prescribing a diagnostic test, taking a diagnostic pregnancy test and/or receiving the results of any such test or examination. Pregnancy status to "verify" the pregnancy status of female subjects. Illustrative embodiments

In one embodiment, the present disclosure provides 2-{3-[4-(1H-indazol-5-ylamine)-2-quinazolinyl]phenoxy}-N-(propan-2- acetamide or its pharmaceutically acceptable salt ( compound ) or its mesylate (berusudil) for the treatment of cGVHD in certain subpopulations of patients who are reproductive risk patients.

In one embodiment, the reproductive risk (RR) patient is a female patient of reproductive potential; in another embodiment, the RR patient is a male patient of reproductive potential whose female partner; in another embodiment, The RR patient is a lactating patient; and in another embodiment, the RR patient is a pregnant patient.

In some embodiments, the RR patient is a female patient of reproductive potential who uses effective contraception during treatment; in some embodiments, the female patient uses effective contraception during treatment and for at least one week after the last dose of the compound. Effective contraception; in some embodiments, the RR patient is a male patient of reproductive potential who uses effective contraception during treatment; in some embodiments, the male patient uses effective contraception during treatment and at least after the last dose of the compound Use effective contraception within one week;

In one embodiment, the present disclosure provides use of a compound or a mesylate salt thereof (berusudil) in the treatment of cGVHD in a patient with RR, comprising informing the patient to self-treat with the compound or berusudil Reproductive risks to the fetus or embryo and/or to the breastfed child. In another embodiment, the present disclosure provides for verifying the status of a RR patient (e.g., in the case of a female, whether she is pregnant) prior to administration of the compound or berusudil.

In another embodiment, the present disclosure provides the use of a compound or its mesylate (berusudil) in the treatment of cGVHD in a female patient of reproductive potential, comprising advising the patient during treatment and during Use effective contraception for at least one week after the last dose of compound.

In another embodiment, the present disclosure provides use of a compound, or a mesylate salt thereof (berusudil), in the treatment of cGVHD in a female patient who is pregnant or pregnant at the time of taking the compound, comprising informing the female There is a potential risk to the fetus in patients receiving treatment with the compound while pregnant.

In some embodiments, a patient being treated for cGVHD may become pregnant while receiving treatment with a compound. In the embodiments, the present disclosure contemplates informing the female patient of the potential risks to the fetus if she is pregnant and receiving treatment with a compound.

In some embodiments, the present disclosure provides use of a compound or a mesylate salt thereof (berusudil) in the treatment of cGVHD in a male patient with a female partner of reproductive potential, comprising advising the male patient on the use of the compound Use effective contraception during treatment and for at least one week after the last dose of compound.

In another embodiment, the present disclosure provides the use of a compound or its mesylate (berusudil) in the treatment of cGVHD in a lactating patient, comprising advising the patient during and at the end of treatment with the compound No breastfeeding should be performed for at least one week after a dose of the compound.

In another embodiment, the present disclosure provides the use of a compound or its mesylate salt (berusudil) in the treatment of cGVHD in a patient at risk for pregnancy who is taking the compound or berusudil Effective contraception is being used during treatment. In some embodiments, the pregnancy-risk patient is a female; in other embodiments, the pregnancy-risk patient is a male. In another embodiment, the pregnancy-risk patient uses effective contraception during treatment and for at least one week after the last dose of the compound.

In another embodiment, the present disclosure provides use of a compound, or its mesylate salt (berusudil), in the treatment of cGVHD in a non-lactating patient.

In some embodiments, the compound or its mesylate salt (berusudil) is administered to the patient at a dose of 200 mg per day.

The present disclosure further provides methods of treating cGVHD in a patient with a compound or a mesylate thereof (berusudil), comprising the steps of: (a) verifying whether the patient is a reproductive risk patient; and (b) ( i) if it is verified that the patient is not a patient at reproductive risk, administer berusudil to the patient, or (ii) if the patient is verified to be a patient at reproductive risk, inform the patient to receive berusudil Potential reproductive risks of treatment with lusudil, and/or the use of effective contraception is recommended during treatment with the compound and for at least one week after the last dose of the compound; and/or in the case of lactating patients, the use of effective contraception is recommended during treatment with the compound Do not breastfeed during and for at least one week after the last dose of compound.

In some embodiments, the patient can be verified to be a lactating patient; or a pregnant patient; or a male or female pregnancy risk patient. In such embodiments, the present disclosure contemplates informing the patient of the reproductive risks from berusudil; and/or advising on the use of effective contraception during treatment with the compound and for at least one week after the last dose of the compound; and /or where applicable, it is recommended not to breastfeed during treatment with berusudil and for at least one week after receiving its last dose.

In some embodiments, the present disclosure provides a method of treating cGVHD in a patient, comprising: (a) verifying whether the patient is a reproductive risk patient; and (b) (i) if it is verified that the patient is not a reproductive risk patient, then administer berusudil to said patient, or (ii) if said patient is verified to be a reproductive risk patient, during treatment with berusudil and for at least one week after receipt of the last dose Use effective contraception.

In some embodiments, the subject (or patient) has undergone an allogeneic hematopoietic stem cell transplant as a matched HSCT. In some embodiments, the allogeneic hematopoietic stem cell transplant is haploidentical HSCT.

In some embodiments, berusudil treatment is continued based on patient tolerance until active cGVHD symptoms resolve or progress. The number and duration of treatment depends on the patient. In some embodiments, berusudil is administered to the patient in one or more 28-day cycles.

In some embodiments, the number of cycles ranges from 3 to 15. In some embodiments, the number of cycles ranges from 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5 Or 3 to 4. In some embodiments, the number of cycles ranges from 5 to 11. In some embodiments, the number of cycles ranges from 6 to 12. In some embodiments, the number of cycles ranges from 5 to 10, 5 to 9, or 5 to 8. In some embodiments, the number of cycles ranges from 5 to 7. In some embodiments, the number of cycles ranges from 5 to 6. In some embodiments, the number of cycles is five. In some embodiments, the number of cycles is six. In some embodiments, the number of cycles is seven. In some embodiments, the number of cycles is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

In some embodiments, the subject has chronic graft-versus-host disease and has experienced failure of one to three prior lines of systemic therapy for chronic graft-versus-host disease. In some embodiments, the subject has chronic graft-versus-host disease and has experienced failure of at least two prior lines of systemic therapy for chronic graft-versus-host disease. In some embodiments, the subject has chronic graft-versus-host disease and has experienced failure of two to five prior lines of systemic therapy for chronic graft-versus-host disease. In another embodiment, the subject has failed at least once, at least twice, at least three times, at least four times, or at least five times.

In some embodiments, the subject experienced a complete response to the last treatment for graft-versus-host disease prior to berusudil. In some embodiments, the subject experienced a partial response to the last treatment for graft-versus-host disease prior to begrusudil. In some embodiments, the subject experienced stable disease during the last graft-versus-host disease treatment prior to berusudil.

In some embodiments, a prior line of systemic therapy for chronic graft-versus-host disease has been discontinued.

In some embodiments, the prior line of systemic therapy is selected from the group consisting of prednisone, tacrolimus, ECP, sirolimus, ibrutinib, ruxolitinib, MMF, rituximab, MTX, cyclosporine , imatinib, ixazomib, and ofatumumab.

In some embodiments, the cGVHD is steroid-refractory (SR) cGVHD. In some embodiments, the subject is refractory to the last line of treatment prior to berusudil treatment.

In some embodiments, the subject is receiving concomitant corticosteroid therapy. In some embodiments, the concomitant corticosteroid therapy is selected from the group consisting of prednisone, prednisolone, methylprednisolone, and budesonide. In some embodiments, the concomitant corticosteroid therapy is prednisone. In some embodiments, the dose of concomitant corticosteroid therapy is reduced after at least 1 cycle of berusudil treatment. In some embodiments, the dose of concomitant corticosteroid therapy is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70%. In some embodiments, the dose of concomitant corticosteroid therapy is reduced after at least 1 cycle of berusudil treatment by at least about 10% to about 70%, about 15% to about 65%, about 20% to about 60%, about 30% to about 60%, about 35% to about 60%, about 40% to about 60%, or about 45% to about 55%. In some embodiments, concomitant corticosteroid therapy is discontinued after at least 1 cycle of berusudil treatment.

In some embodiments, the subject is receiving concomitant calcineurin inhibitor therapy. berusudil tablets

In one embodiment, berusudil is formulated as a lozenge for oral administration. Begrusudil mesylate is a yellow powder that is nearly insoluble in water. Begrusudil tablets may be prepared for oral administration. Each lozenge contains 200 mg free base, equivalent to 242.5 mg berusudil mesylate. Tablets may also contain the following inactive ingredients: microcrystalline cellulose, hypromellose, croscarmellose sodium, colloidal silica, and magnesium stearate. The tablet film consists of polyvinyl alcohol, polyethylene glycol, talc, titanium dioxide and yellow iron oxide. Each 200 mg tablet is a pale yellow film-coated, oval-shaped tablet with a debossed pattern of "KDM" on one side and a debossed pattern of "200" on the other side. Store tablets at room temperature 20ºC to 25ºC (68°F to 77°F); short-term travel allowed 15ºC to 30ºC (59°F to 86°F).

The following abbreviations may be helpful in considering the description of this article. Abbreviation AE adverse events AMS accelerator mass spectrometry alloHCT allogeneic hematopoietic cell transplantation BID twice daily (twice daily) BM marrow ikB chronic graft versus host disease CMV cytomegalovirus CR full response DDI drug-drug interactions DOR reaction duration EOI end of infusion FFS No failure survival GD pregnancy day HLA human leukocyte antigen IST immunosuppressive therapy IV intravenously LSC liquid scintillation counting LSS Lee Symptom Scale NMT no more than NOAEL No observable adverse effect level ORR overall response rate OS overall survival PBSC peripheral blood stem cells PR partial reaction QOL quality of life SD standard deviation SR Steroid refractory TEAE Adverse events that occurred during treatment TK toxicokinetics TTNT Time to next treatment QD every day; every day Examples Example 1 : Preliminary Prenatal Developmental Toxicity and Toxicokinetic Studies in Sprague-Dürer Rats Study Objectives and Design

This pilot study was conducted to provide information on berusudil dose selection for use in subsequent embryo-fetal developmental toxicity studies in Sprague-Dulle rats (Example 3). The second part of this study was a preliminary developmental toxicity study, i.e., when berusudil was administered once daily to pregnant rats by oral gavage from gestation day (GD) day (GD) 17, the Toxicokinetics of berusudil and its two metabolites KD025m1 and KD025m2. The design of the study is shown in Table 1. Table 1 : Example 1 Dosage Preliminary/Design of Rat Study group treatment Dose level (mg base /kg) Dosage volume (mL/kg) Dose concentration (mg base /kg) Number of animals initial uterine examination toxicokinetics F F F 1 control 0 10 0 5 5 - 2 low dose 25 10 2.5 5 5 - 3 low-medium dose 50 10 5.0 5 5 - 4 medium-high dose 150 10 15.0 5 5 - 5 high dose 300 10 30.0 5 5 - 6 control 0 10 0 3 - 3 7 low dose 25 10 2.5 6 - 6 8 low-medium dose 50 10 5.0 6 - 6 9 medium-high dose 150 10 15.0 6 - 6 10 high dose 300 10 30.0 6 - 6 Total number of animals: 52 25 27

A total of 52 Sprague-Dürer (SD) rats were used in this study. Vehicle ( 0.4% (w/w) methylcellulose (400 cps) in distilled water or berusudil was administered once daily to regularly mated female SD rats. Assign animals to groups as shown in Table 1. observe

Observation of animals includes cageside assessment (twice daily), evaluation of clinical signs (daily from GD 6 to 20), body weight (on arrival - GD 2 or 3 - and GD 6, 9, 12, 15, 18 and 20) , food intake (GD 6-20) and anatomic pathology including uterine examination. Toxicokinetic assessment of berusuudil and metabolites. All fetuses were examined externally.

Clinical signs are limited to 300 mg base/kg/day and have a low incidence but are considered potentially related to treatment. Clinical signs consist of an unkempt and/or emaciated appearance and a hunched posture.

Mean body weight gain and food intake at 25 and 50 mg base/kg/day decreased slightly (dose-related) from GD 6-9, GD 6-18 and GD 6-20. At 150 mg base/kg/day, mean body weight decreased throughout gestation, with weight loss occurring from GD 6-9 and reduced weight gain noted from GD 12-15, GD 6-18, and GD 6-20. During these same intervals, food intake decreased correspondingly at 150 mg base/kg/day. Increases in body weight and food intake after treatment at 25, 50 and 150 mg base/kg/day (GD 18-20) are considered rebound/recovery effects. Following initiation of dosing at 300 mg base/kg/day on GD 6, mean body weight values, mean body weight gain and food intake were adversely affected at each time interval.

No adverse maternal autopsy findings were noted in any treatment group. The number of litters available for examination was 5 in each of the 0, 25, 50, 150 and 300 mg base/kg/day groups. At dose levels of 25, 50 and 150 mg base/kg/day, all females maintained pregnancy until the scheduled necropsy day on GD 20.

There was no apparent dose response in the in utero data at 25, 50, and 150 mg base/kg/day, but the mean number of early resorptions and the mean number of viable fetuses was significantly increased in the 50 mg base/kg/day group. slightly reduced. The average post-implantation losses (%/litter) at 0, 25, 50, 150 and 300 mg base/kg/day were 4.41, 4.00, 11.62, 4.51 and 24.51 respectively. The average surviving litter sizes at these same dose levels were 12.8, 13.2, 11.6, 12.2 and 10.0, respectively.

The mean fetal weights (female fetuses) at 0, 25, 50, 150 and 300 mg base/kg/day were 1.36, 4.19, 4.39, 4.18 and 3.34 g respectively. The combined mean fetal weights (males and females) at these same dose levels were 4.50, 4.37, 4.50, 4.22 and 3.42 g, respectively. At 300 mg base/kg/day, post-implantation losses (early resorption) were increased (including in females with total embryonic loss), and mean fetal weight was significantly reduced.

Although the historical incidence of external fetal anomalies in studies found to be atypical for the dose range in rats, litter incidence in this study was significant, ranging from 40 to 40 at 50 and 150 mg base/kg/day, respectively. % and 20% of the litters were affected, and 25% of the litters were affected at 300 mg base/kg/day. In the 50 mg base/kg/day group, one fetus was edematous (all over the body), and another fetus/litter in this dose group had no anus and tail. In the 150 mg base/kg/day group, one fetus had an omphalocele. At 300 mg base/kg/day, a fetus has a dome-shaped head. result

In this oral preliminary prenatal developmental toxicity study with berusudil, maternal toxicity was considered mild at 25 and 50 mg base/kg/day and moderate at 150 mg base/kg/day. (Moderate is defined as more prominent, with a significant potential for increased severity. Limitation of tissue or organ function is possible.) Maternal, embryonic, and fetal toxicity is considered excessive at 300 mg base/kg/day and is Selection in subsequent developmental toxicity studies in rats will exclude this dose level.

For the parent compound berusudil, the 25 mg base/kg/day dose level corresponds to a parent C _max of 1800 ng/mL at the end of the dosing regimen (GD 17) and a parent AUC _0-24 of 20400 ng ·hr/mL; for KD025m1, the parent C _max is 176 ng/mL and the AUC _0-24 is 2230 ng·hr/mL; and for KD025m2, the parent C _max is 127 ng/mL and the AUC _0-24 is 1480 ng· hr/mL. Example 2 : Preliminary Prenatal Developmental Toxicity and Toxicokinetic Study in New Zealand White Rabbits with a Non-Pregnant Dose Range Discovery Phase Study Objectives and Design

This study was conducted to provide information for berusudil dose selection for use in a subsequent embryo-fetal developmental toxicity study in New Zealand White (NZW) rabbits (Example 4). The study was conducted in two phases: Phase A was a dose-ranging tolerability study with non-pregnant rabbits, and Phase B was a preliminary developmental toxicity study. For both groups, vehicle (in distilled water) was administered via oral gavage once daily for 5 consecutive days (Phase A) or once daily from gestation day (GD) 6 to 18 (Phase B). 0.4% (w/w) methylcellulose (400 cps)) or berusudil was administered to nonpregnant or regularly mated female NZW rabbits. The design of the study is further set out in Table 2 below. Table 2 : Design of Dosing Preliminary/Rabbit Study for Example 2 Table 2 : Research design Group number Dosage level (mg base/kg/day) number of females Stage A (not pregnant) ^a 1/5 25/100 2 2/6 3/7 50/200 75/ ^300b 2 2b 4/8 125/400 2 Phase B Main Research (Timed Mating) 9 0 6 10 25 6 11 50 6 12 100 6 13 250 6 Phase B toxicokinetics (timed mating) 14 0 4 15 25 4 16 50 4 17 100 4 18 250 4 ^a The test article is administered to the animals for 5 consecutive days, followed by a 10-day washout period, and then the test article is administered at a higher dose level for 5 consecutive days; ^b The dose levels appear to be ascending doses; however, after the first dose Replacement of a female (Animal No. 105). As a result, only replacement animals were administered the second dose (300 mg base/kg/day); the total number of animals studied in Phase A was 9. observe

Observations of animals included clinical signs, body weight, food intake, and anatomical pathology including uterine examination. Toxicokinetic evaluation of berusuudil and two metabolites. Give all fetuses an appropriate external examination.

Survival was not affected at any dose level administered in Phase A. All animals survived until the scheduled termination date. In Phase A, clear treatment-related effects were seen at dose levels of 300 and 400 mg base/kg/day and consisted of mild weight loss and reduced food intake.

Potential treatment-related clinical findings were seen on the final day of the study (Study Day 20) at corresponding dose levels of 100, 200, 300, and 400 mg base/kg/day administered from Study Days 15 to 19 Female 108 had abnormal feces (little/none) at 400 mg base/kg/day, with brown discoloration in the anogenital area.

In Phase B, survival was not affected at any dose administered. Dose-dependent maternal effects occurred at the 100 and 250 mg base/kg/day dose levels and consisted of: decreased/reduced weight gain, decreased food intake, clinical signs (loss of appetite, abnormal stools, emaciated appearance) and Overall dose-related increase in the amount of veterinary intervention (food fortification). No treatment-related maternal necropsy findings were observed, and treatment-related embryonic or fetal toxicity was not apparent at any dose level.

result

When berusudil was administered to pregnant rabbits at 25, 50, 100, and 250 mg base/kg/day from gestation days 6 to 18, no observations were observed at 25 or 50 mg base/kg/day. to toxicologically relevant maternal toxicity. This was clearly demonstrated by decreased/reduced weight gain, decreased food intake, clinical signs (loss of appetite, abnormal stool, emaciated appearance) and overall dose-related increases in the amount of veterinary intervention (food fortification) at 100 and 250 mg base/ kg/day dose-dependent maternal response.

At the NOAEL for maternal toxicity (50 mg base/kg/day), for begrusudil, a dose level of 50 mg base/kg/day corresponds to a maternal C _max at the end of the dosing regimen (GD 18) of 621 ng/mL, and the parent AUC _0-24 is 2480 ng·hr/mL; for KD025m1, the parent C _max is 528 ng/mL and the AUC _0-24 is 1320 ng·hr/mL; and for KD025m2, the parent C _max is 1090 ng/mL and AUC _0-24 is 2990 ng·hr/mL. At the NOAEL for developmental toxicity (250 mg base/kg/day), for begrusudil, a dose level of 250 mg base/kg/day corresponds to a maternal C _max at the end of the dosing regimen (GD 18) of 2100 ng/mL, and the parent AUC _0-24 is 15600 ng·hr/mL; for KD025m1, the parent C _max is 1040 ng/mL and the AUC _0-24 is 4760 ng·hr/mL; and for KD025m2, the parent C _max is 3080 ng/mL and AUC _0-24 is 19700 ng·hr/mL.

Based on the results of this study, dose levels of 0, 50, 125, and 225 mg base/kg/day were selected for an embryo-fetal developmental toxicity study in New Zealand white rabbits (Example 4). Example 3 : Embryo-fetal developmental toxicity study with toxicokinetic evaluation in Sprague-Dürer rats Study objectives and design

This study was conducted to determine the embryo-fetal developmental toxicity of berusudil in Sprague-Dulle rats, including teratogenic potential. The study also included toxicokinetic (TK) evaluations to determine the exposure/toxicity relationship for berusuudil and its metabolites (KD025m1 and KD025m2). From gestation day (GD) 6 to 17, vehicle (0.4% (w/w) methylcellulose (400 cps) in distilled water) or berusudil was administered via oral gavage. Administer once daily to regularly mated SD rats.

For this study, a total of 140 regularly mated female rats (approximately 8 to 10 weeks old) were obtained from Charles River Laboratories, Raleigh, NC, and allowed to acclimate from arrival until dosing time on GD 6. During the acclimatization period, animals were observed twice daily for general health and any signs of disease. All animals were given a detailed clinical examination and body weights were recorded before selection. Animals assigned to the study had body weights within ±20% of mean body weight. Food intake was collected during the acclimatization period.

One hundred and thirty-three female animals (weighing 162 g to 226 g, randomized) were assigned to control, treatment, and TK groups using a standard randomization procedure based on weight measurements, as shown in Table 3. Table 3 : Group Allocation for Example 3 Study Table 3 : Group allocation Group number Dosage level (mg base/kg/day) number of females that mate regularly main research 1 0 25 2 15 25 3 50 25 4 150 ^25a toxicokinetics 5 0 3 6 15 10 7 50 10 8 150 10 ^a Five animals (animals 277 and 289 to 292) at 150 mg base/kg/day were excluded from the summary table due to suspicion of gavage injury. invest

Vehicle and berusudil were administered once daily via oral gavage from GD 6 to 17 at approximately the same time each day (± 2 hours from GD 6 dosing). Treatment group dose levels were 15, 50, and 150 mg base/kg/day with a dose volume of 10 mL/kg. The control group received vehicle in the same manner as the treatment group. Additionally, TK animals received vehicle or berusudil in the same manner and at the same dose levels and volumes as the main study group. The vehicle and berusudil formulation are continuously stirred at room temperature for at least 30 minutes prior to and throughout dosing. Individual dosage is based on recent body weight. observe

Animals were evaluated during life and postmortem. Intravital evaluation includes cage-side observations (for morbidity, mortality, injury), detailed clinical signs, body weight, food intake and anatomic pathology including uterine examination. Toxicokinetic evaluation of berusuudil and metabolites (KD025m1 and KD025m2). All fetuses were examined externally and internally or skeletally. Cageside and clinical observations.

No treatment-related deaths were observed at any dose level. Five females treated with 150 mg base/kg/day suffered suspected gavage injuries. In order to discuss the study results clearly, these five females are not included in the list of summarized data.

Detailed clinical observations were performed daily from GD 6 to 20 (4 hours ± 1 hour after dosing the daily dose). Such observations include, but are not limited to, evaluation of the skin, fur, eyes, ears, nose, mouth, chest, abdomen, external genitalia, extremities and feet, and evaluation of breathing. Clinical observations are summarized in Table 4 below. Table 4 : Summary of pregnancy clinical observations from the Example 3 study (days 6 to 20)* Dosage (base/kg/day) Observations 0 mg 15 mg 50 mg 150 mg number of animals observed 25 25 25 20 Appearance discharge, brown, vulva 1/1 1/1 1/1 4/4 discharge, red, vulva 1/1 0/0 4/4 1/1 Ear/missing part of ear, ear/left 0/0 13/1 0/0 15/1 Missing tail - part 0/0 0/0 0/0 11/1 Sparse coat / skin hair, abdominal area 0/0 7/1 0/0 0/0 Thinning hair, neck area 0/0 0/0 8/1 0/0 Thinning hair, forefoot/left 5/1 4/2 0/0 27/3 Sparse hair, forefoot/right 0/0 0/0 0/0 23/3 Sparse hair, forelimb/left 0/0 12/3 10/2 14/2 Sparse hair, forelimb/right 0/0 17/3 10/2 30/3 Sparse hair, hindlimb/left 0/0 0/0 0/0 6/1 Sparse hair, hindlimb/right 0/0 0/0 0/0 6/1 Thinning hair, waist area 0/0 0/0 0/0 12/1 Sparse hair, chest area 0/0 0/0 7/1 12/2 scabbed areas, face 0/0 7/1 0/0 0/0 Scab area, forelimb/right 0/0 4/1 0/0 0/0 scab area, tail 0/0 0/0 0/0 13/2 scab area, chest area 0/0 1/1 0/0 0/0 • Number of observations/total animal weight.

Measure and record the body weight of all animals on GD 6, 9, 12, 15, 18 and 20. Individual body weight changes were calculated for the following GD intervals: 6-9, 9-12, 12-15, 15-18, 18-20, 6-18 and 6-20. Adjusted body weight (GD 20 weight minus gestational uterine weight) and adjusted body weight change (GD 6 to 20) were also calculated.

Maternal weight and weight changes are summarized in Tables 5 and 6 below: Table 5 : Maternal body weight ( g ) Group ( mg base /kg/ day) 0 15 50 150 GD 9 236.4 231.7 225.5 ^{a 215.8b} GD 12 262.3 257.0 ^{246.3b 232.1b} GD 15 279.3 272.8 ^{262.6b 244.0b} GD 18 320.7 315.5 ^{298.1b 275.3b} GD 20 355.8 349.6 ^{335.2b 317.0b a} is significantly different from the control: p ＜ 0.05 ^b is significantly different from the control: p ＜ 0.01 Table 6 : Maternal weight change ( g ) Group ( mg base /kg/ day) 0 15 50 150 GD 6-9 13.3 10.0 ^6.2b -4.2 ^b GD 6-18 97.6 93.8 ^{78.8b 55.4b} GD 6-20 132.7 127.9 ^{115.9b 97.1b} GD 18-20 35.1 34.0 37.1 ^{41.8b s} significantly different from control: p < 0.01

Mean maternal body weight decreased statistically significantly in a dose-related manner at 50 and 150 mg base/kg/day throughout the treatment period and in the post-treatment period (GD 9, 12, 15, 18, and 20). at 50 mg base/kg/day and

Mean body weight gain was also reduced in a dose-related manner at 150 mg base/kg/day; from GD 6-9 and 9-12, the reduction in body weight gain was statistically significant at 50 and 150 mg base/kg/day. From GD 12-15, body weight gain at 50 mg base/kg/day was comparable to the control group, but body weight gain at 150 mg base/kg/day remained significantly lower during this same time period. During the last few days of the treatment period (GD 15-18), weight gain remained slightly (statistically significant) reduced at both dose levels, and after the treatment period (GD 18-20), mean weight gain increased were similar (50 mg base/kg/day) or greater (150 mg base/kg/day; statistically significant) than controls, indicating a reinstatement response. Evaluation of the combined intervals (GD 6-18 and 6-20) revealed consistent dose-related reductions in mean maternal body weight gain at 50 and 150 mg base/kg/day (statistically significant at both dose levels ). Food intake.

Food intake of the primary study animals was measured and recorded on corresponding weight days and calculated for the same intervals. Mean maternal food intake was dose-related at 50 and 150 mg base/kg/day throughout the treatment period (GD 6-9, 9-12, 12-15, 15-18, 6-18, and 6-20) in a statistically significant manner. After the treatment period (GD 18-20), food intake was similar to that in the control group, indicating a reinstated response.

Maternal food intake is summarized in Table 7 below: Table 7 : Maternal food intake / % difference from control Group ( mg base /kg/ day) 0 15 50 150 GD 6-9 20.3 19.0 ^{15.5b 10.0b} GD 6-18 23.7 22.6 ^{20.0b 15.4b} GD 6-20 24.3 23.2 ^{21.3b 17.4b} GD 18-20 27.9 27.3 29.0 29.4 ^{bSignificantly} different from control: p < 0.01 Postmortem examination of uterus, ovaries and fetus

All animals in the study were pregnant, resulting in 100% pregnancy index in each study group. The number of litters available for examination was 25, 25, 25 and 20 in the control group, 15 mg alkali/kg/day group, 50 mg alkali/kg/day group and 150 mg alkali/kg/day group respectively.

On GD 20, each surviving primary study female was euthanized by carbon dioxide inhalation, followed by intraperitoneal venous exsanguination and immediate caesarean section. The skin is turned over through the ventral midline incision to examine the breast tissue and locate any subcutaneous mass. The abdominal cavity is then opened, and the uterus is exposed. The uterus was removed, and the gravid uterine weight was recorded. Starting from the distal end of the left uterine horn, the positions of viable and nonviable fetuses, early and late resorptions for each uterine horn, and the total number of implantations were recorded. The number of corpus luteum on each ovary was also recorded.

The fetus is removed by making a dorsal incision longitudinally along the two uterine horns. The embryonic membranes of each fetus are gently removed and each fetus is separated from the placenta, allowing the umbilical cord to be fully stretched. Examine the placenta roughly.

Appearing non-pregnant uteri from females were opened and placed in 10% ammonium sulfide solution to detect implantation sites. If a lesion is detected, it is considered to be an early resorption and data from this female are included in the average calculation

At 150 mg base/kg/day, mean gestational uterine weight was slightly reduced. However, at 150 mg base/kg/day, mean final body weight, adjusted final body weight, and adjusted final body weight change were statistically significantly reduced when compared to corresponding control values and were considered treatment relevant.

Mean gestational uterine weight at 50 mg base/kg/day was similar to control values. However, at 50 mg base/kg/day, mean final body weight, adjusted final body weight, and adjusted final body weight change were statistically significantly reduced when compared to corresponding control values and were considered treatment relevant.

Gestational uterine weight and adjusted body weight/weight change are summarized in Table 8. Fetal weights are summarized in Table 9. Table 8 : Summary of gravid uterine weight and adjusted body weight / weight change values 0 mg base/kg/day (N=25) 15 mg base/kg/day (N=25) 50 mg base/kg/day (N=25) 150 mg base/kg/day (N=20) end point average value SD average value SD average value SD average value SD Pregnant uterus weight, g 69.3 9.15 71.0 9.59 68.8 8.42 64.3 10.54 Final weight, g 355.8 24.12 349.6 22.81 ^335.2b 26.51 ^317.0b 21.49 Adjusted final weight, g 286.5 19.52 278.5 19.88 ^266.4b 23.14 ^252.7b 15.85 Adjusted weight change relative to day 6 63.4 11.68 56.8 11.83 ^47.1b 10.64 ^32.8b 12.59 N = number of measurements used to calculate mean SD (standard deviation) b = significantly different from control (p < 0.01) Table 9 : Summary of mean fetal weight ( g ) / % difference from control Group ( mg base /kg/ day) 0 15 50 150 Male fetal weight 4.25 4.19 4.19 ^3.91b /-8.0% Female fetal weight 4.03 4.00 3.94 ^3.72b /-7.7% Combined gender fetal weight 4.12 4.10 4.06 ^3.82b /-7.3% ^{bSignificantly} different from control: p < 0.01

For subjects treated with 15 and 50 mg base/kg/day, none of the fetal weight values were statistically significant when compared to the corresponding control values. At 150 mg base/kg/day, all mean fetal weight values for males, females and combined sexes were statistically significantly reduced; these values were reduced by between -7% and -8% compared to the corresponding control values. Toxicokinetic analysis

Exposure to berusudil, KD025m1, and KD025m2 increased as berusudil dose levels increased from 15 mg base/kg to 150 mg base/kg. The increase in berusudil C _max in pregnant rats was generally less than dose proportional at GD 6 and GD 17, and the increase in AUC _0-24 was generally dose proportional at GD 6 and GD 17. Increases in KD025m1 C _max values in pregnant rats were generally dose proportional at GD 6 and GD 17, and increases in AUC _0-24 were greater than dose proportional at GD 6 and GD 17. Increases in KD025m2 C _max and AUC _0-24 values in pregnant rats were generally dose-proportional at GD 6 and greater than dose-proportional at GD 17. No significant accumulation of begrusudil, KD025m1, and KD025m2 was observed after multiple administrations of berusuudil in pregnant rats. AUC _0-24 metabolite to maternal ratios indicate that berusuudil is converted to KD025m1 and KD025m2 in pregnant rats following oral gavage administration. For KD025m1 and KD025m2, the metabolite to parent ratio ranged from 0.0799 to 0.188 and 0.0482 to 0.125, respectively.

These TK results are summarized in Table 10 below. Table 10 : Summary of mean berusudil, KD025m1 , KD025m2 _Cmax and AUC _0-24 in plasma of pregnant rats Gap (GD) dose group Dosage level (mg base /kg) KD025 KD025m1 KD025m2 C _max (ng/mL) AUC _0-24 (ng*hr/mL) C _max (ng/mL) AUC _0-24 (ng*hr/mL) C _max (ng/mL) AUC _0-24 (ng*hr/mL) 6 6 15 1530 9750 152 978 76.2 515 7 50 3000 37700 349 5040 191 3050 8 150 5150 89400 955 16400 519 10500 17 6 15 1320 11900 93.3 948 55.6 572 7 50 4360 33300 434 4780 350 3580 8 150 7800 88300 1310 16600 972 11100 result

When berusudil was administered to pregnant rats at 15, 50, and 150 mg base/kg/day from gestation days 6 to 17, maternal and fetal development occurred at 150 mg base/kg/day. Toxicity; reductions in mean maternal body weight, body weight gain, and food intake were significant. Mean fetal weight was also significantly reduced and considered unfavorable. The 50 mg base/kg/day dose level resulted in adverse dose-related reductions in mean maternal body weight, weight gain, and food intake. Dosage levels of 15 mg base/kg/day did not produce any adverse maternal or fetal developmental effects. The dose level of 50 mg base/kg/day is considered the NOAEL for fetal developmental toxicity.

For the parent compound berusudil, a dose level of 50 mg base/kg/day corresponds to a parent C _max of 4360 ng/mL at the end of the dosing regimen (GD 17) and a parent AUC0-24 of 33300 ng· hr/mL; for KD025m1, the parent C _max is 434 ng/mL and the AUC 0-24 is 4780 ng·hr/mL; and for KD025m2, the parent C _max is 350 ng/mL and the AUC _0-24 is 3580 ng·hr/ mL. A dose level of 15 mg base/kg/day is considered the NOAEL for maternal toxicity. For the parent compound berusudil, the 15 mg base/kg/day dose level corresponds to a parent C _max of 1320 ng/mL at the end of the dosing regimen (GD 17) and a parent AUC _0-24 of 11900 ng hr/mL; for KD025m1, the parent C _max was 93.3 ng/mL and the AUC _0-24 was 948 ng hr/mL; and for KD025m2, the parent C _max was 55.6 ng/mL and the AUC _0-24 was 572 ng · hr/mL. Example 4 : Embryo-fetal developmental toxicity study with toxicokinetic evaluation in New Zealand White Rabbits

The purpose of this study was to determine the embryo-fetal developmental toxicity, including teratogenic potential, of berusudil in New Zealand White (NZW) rabbits. The study also included toxicokinetic evaluations to determine the exposure/toxicity relationship for berusuudil and metabolites (KD025m1 and KD025m2). From gestation day (GD) 6 to 18, vehicle (0.4% (w/w) methylcellulose (400 cps) in distilled water) or berusudil was administered via oral gavage. Administer once daily to regularly mated female New Zealand White (NZW) rabbits. 111 female animals (weight 2.64 to 3.56 kg, randomized) were assigned to the control, treatment and TK groups identified in Table 11 below using a standard randomization procedure by weight measurements. Table 11. Design of the Example 4 Study Group number Dosage level (mg base/kg/day) number of females that mate regularly main research 1 0 twenty three 2 50 twenty three 3 125 twenty three 4 225 twenty three toxicokinetics 5 0 4 6 50 5 7 125 5 8 225 5

Animals assigned to the study had body weights within ±20% of mean body weight. Animals were housed individually in suspended stainless steel cages in an environmentally controlled room. Provide animal nutrition fortification according to SOP. Fluorescent lighting is provided for approximately 12 hours per day. Interrupt the dark cycle intermittently due to study-related activity. Monitor, record, and maintain temperature and humidity within the ranges of 61 to 72°F and 30 to 70%, respectively, to the greatest extent possible. Food was provided from 08:00 to 12:00 in the morning starting on the second day of acclimatization and continued throughout the study. During the dosing period, food is offered 1.5 hours ± 30 minutes before dosing and maintained as needed until the next food offer. invest

Vehicle and berusudil were administered once daily via oral gavage from GD 6 to 18 at approximately the same time each day (at GD 6, ± 2 hours from the first dose). The dose levels for the treatment groups were 50, 125, and 225 mg base/kg/day, and the dose volume was 10 mL/kg. The control group received vehicle in the same manner as the treatment group. Additionally, TK animals received vehicle or berusudil in the same manner and at the same dose levels and volumes as the main study group. The vehicle and berusudil formulation are continuously stirred at room temperature for at least 30 minutes prior to and throughout dosing. Individual dosage is based on recent body weight. observe

Observations of animals included clinical signs, body weight, food intake, and anatomical pathology including uterine examination. Toxicokinetic evaluation of berusuudil and metabolites (KD025m1 and KD025m2). External, visceral, and skeletal examinations were performed on all fetuses. Cageside and clinical observations

All animals were observed cageside twice daily (for morbidity, death, injury). Every day from GD 6 to 29 (4 hours ± 1 hour after daily dose), each primary study animal was removed from the cage and given a detailed clinical examination. Sometimes, clinical observations are recorded at irregular intervals. Such observations include, but are not limited to, evaluation of the skin, fur, eyes, ears, nose, mouth, chest, abdomen, external genitalia, extremities and feet, and evaluation of breathing. Two animals aborted on GD 19, respectively at 125 and 225 mg base/kg/day (animal numbers 251 and 282, respectively). One additional animal at 225 mg base/kg/day was found dead on GD 9. These events are considered adverse and treatment-related.

Clinical findings of body weight loss were seen in the 225 mg base/kg/day group and may be related to the decreases seen in weight gain and food intake data that are considered treatment-related and undesirable. For the two animals that aborted at 125 and 225 mg base/kg/day, red material was seen in the cage tray and the abortions were considered treatment-related and undesirable (see previous section).

Maternal survival and pregnancy status are summarized in Table 12 below. Clinical observations are summarized in Table 13 below. Table 12 : Main study maternal survival Group ( mg base /kg/ day) 0 50 125 225 Number of females in the study twenty three twenty three twenty three twenty three number of non-pregnancies 0 1 3 0 Number of abortions 0 0 1 1 Number of deaths 0 0 0 1 GD29 has all absorbed quantities 0 0 0 2 number of surviving litters GD29 fetus twenty three twenty two 19 19 Table 13 : Summary of clinical observations in pregnancy, days 0 to 29 (Example 4)* Dosage (base/kg/day) Observations 0 mg 50 mg 125 mg 225 mg number of animals observed twenty three twenty three twenty three twenty three Animal Husbandry Missing Toenail, Forefoot/Left 0/0 1/1 0/0 0/0 Decreased behavior / activities 0/0 0/0 0/0 4/1 Passing little/no stool 3/2 0/0 0/0 0/0 Contents in pan/litter, red 0/0 0/0 2/1 2/2 Appearance of swelling in nose/nasal opening 2/1 0/0 0/0 0/0 swelling, vulva 0/0 3/1 0/0 2/1 thin 0/0 0/0 0/0 38/5 Coat / skin hair discoloration, brown, anogenital area 0/0 0/0 7/1 0/0 Hair discoloration, red, anogenital area 0/0 0/0 1/1 0/0 Hair discoloration, brown, ear/left 0/0 12/1 0/0 0/0 Hair discoloration, brown, ear/right 0/0 12/1 0/0 0/0 Hair discoloration, yellow, ear/right 5/1 0/0 0/0 0/0 Hair discoloration, brown, facial 4/1 24/3 0/0 12/2 Hair discoloration, redness, face 1/1 0/0 2/1 0/0 Hair discoloration, yellow, facial 0/0 27/2 10/1 14/2 Hair discoloration, red, forelimb/left 0/0 1/1 0/0 0/0 Hair discoloration, brown, hind limb/left 0/0 3/1 0/0 1/1 Hair discoloration, red, chest area 0/0 1/1 0/0 0/0 Sparse hair, anal area 0/0 0/0 15/2 0/0 Sparse hair, armpit area/left 0/0 0/0 2/1 3/1 Thinning hair, armpit area/right 0/0 0/0 2/1 2/1 Thinning hair, neck area 0/0 0/0 1/1 0/0 Sparse hair, ear/left 13/1 0/0 0/0 0/0 Sparse hair, ear/right 4/1 0/0 0/0 0/0 Thinning hair, forefoot/left 0/0 5/1 0/0 3/1 Sparse hair, forefoot/right 0/0 4/1 0/0 5/2 Sparse hair, forelimb/left 0/0 5/1 4/1 0/0 Sparse hair, hindlimb/left 0/0 5/1 0/0 8/1 Thinning hair, groin area/right 0/0 0/0 4/1 0/0 Thinning hair, shoulder/left 0/0 0/0 2/1 0/0 Sparse hair, chest area 0/0 0/0 2/1 1/1 Sparse hair, ventral side 0/0 0/0 19/1 0/0 Scab area, ear/left 7/1 0/0 0/0 0/0 Crusted area, nose/nasal opening 9/2 0/0 0/0 0/0 Skin discoloration, pallor, all over the body 0/0 0/0 0/0 11/1 *Number of observations/total number of affected animals. weight.

Measure and record the body weight of all animals on GD 0, 6, 10, 13, 16, 19, 21, 25 and 29. Individual weight changes were calculated for the following GD intervals: 0-6, 6-10, 10-13, 13-16, 16-19, 19-21, 21-25, 25-29, 6-19, 19-29 and 0 -29. Adjusted body weight (GD 29 weight minus gestational uterine weight) and adjusted body weight change (GD 0 to 29) were also calculated. Record the individual body weight values of TK animals.

No adverse treatment-related effects were seen on body weight data (mean body weight and mean body weight change) at 50 mg base/kg/day. At GD 19, at 125 mg base/kg/day, there was no statistically significant difference and the maximum percentage difference (-5.1%) when comparing mean body weight values to the control group.

However, there was a trend toward lower mean body weight gain during the treatment period at 125 mg base/kg/day. The following differences were statistically significant; mean weight loss occurred from GD 6-10, and mean weight gain decreased from GD 13-16, resulting in an overall reduction in mean weight gain during the treatment period (GD 6-19). After the treatment period at 125 mg base/kg/day, mean body weight gain increased in a statistically significant manner from GD 25-29, resulting in an overall mean body weight gain from GD 19-29 after treatment.

For the entire study period (GD 0-29), body weight gain remained reduced at 125 mg base/kg/day compared with the corresponding control values; the difference with the control group was -8.6%. At 225 mg base/kg/day, mean body weight decreased in a statistically significant manner throughout the treatment period and initially after the treatment period (GD 10, 13, 16, 19, and 21); the range of differences from the control group was -6.3 % to -9.2%. When the mean body weight changes were evaluated at 225 mg base/kg/day, the following differences were statistically significant; mean weight loss occurred from GD 6-10, and mean weight gain decreased from GD 10-13 and GD 13-16, Resulting in overall mean weight loss during the treatment period (GD 6-19). However, there was a trend towards increased mean body weight gain after treatment with 225 mg base/kg/day when compared to the control group; mean body weight gain increased from GD 19-21 and GD 21-25 when compared to the control group. High was statistically significant, resulting in an increase in overall mean body weight in the post-treatment period (GD 19-29). Body weight gain remained reduced (statistically significant) at 225 mg base/kg/day for the entire study period (GD 0-29); the difference compared to the control group was -24.2%.

For the entire treatment period, there was a trend toward reduced weight gain at 125 and 225 mg base/kg/day, which was associated with reduced food intake and was considered an adverse dose-related reaction to berusudil. Trends in increased mean body weight and food intake after treatment at 125 and 225 mg base/kg/day were considered indicative of a rebound/recovery effect.

Maternal weight and weight changes are summarized in Tables 14 and 15 below: Table 14. Maternal body weight ( g ) / % difference from control Group ( mg base /kg/ day) 0 50 125 225 GD 10 3.320 3.316 3.232 ^3.110b /-6.3% GD 13 3.383 3.370 3.262 ^3.111b /-8.0% GD 16 3.463 3.461 3.282 ^3.144b /-9.2% GD 19 3.524 3.493 3.346/-5.1% ^3.227b /-8.4% GD 21 3.575 3.531 3.420 ^3.334a /-6.7% ^a is significantly different from the control: p ＜ 0.05 ^b is significantly different from the control: p ＜ 0.01 Table 15. Maternal weight change ( g ) / % difference from control Group ( mg base /kg/ day) 0 50 125 225 GD 6-10 0.043 0.030 ^{-0.036b -0.160b} GD 6-19 0.247 0.207 ^{0.079b -0.041b} GD 19-29 0.138 0.175 ^{0.241b 0.294b} GD 0-29 0.607 0.592 0.555/-8.6% ^0.460b /-24.2% ^a is significantly different from the control: p ＜ 0.05 ^b is significantly different from the control: p ＜ 0.01 food intake

When comparing food intake at 50 mg base/kg/day to the control group for the entire treatment period GD 6-19, the percent difference was -8.8%. At 125 mg base/kg/day, food intake decreased throughout the treatment period; the following differences were statistically significant; food intake decreased from GD 6-10, 10-13, 13-16, and 16-19 throughout treatment Reduced food intake during the period (GD 6-19).

For the total treatment period (GD 6-19) at 125 mg base/kg/day, the percent difference from the control group was -34.1%. After the treatment period at 125 mg base/kg/day, food intake increased in a statistically significant manner from GD 21-25 and GD 25-29, resulting in an overall increase in food intake from GD 19-29 after treatment. Food intake remained reduced (statistically significant) at 125 mg base/kg/day for the entire study period (GD 0-29); the difference compared to the control group was -11.6%.

At 225 mg base/kg/day, food intake decreased throughout the treatment period; the following differences were statistically significant; food intake decreased from GD 6-10, 10-13, 13-16, and 16-19 throughout treatment Reduced food intake during the period (GD 6-19). For the total treatment period (GD 6-19) at 225 mg base/kg/day, the percent difference from the control group was -49.7%. After the treatment period at 225 mg base/kg/day, food intake increased in a statistically significant manner from GD 21-25 and GD 25-29, resulting in an overall increase in food intake from GD 19-29 after treatment. Food intake remained reduced (statistically significant) at 225 mg base/kg/day for the entire study period (GD 0-29); the difference from the control group was -17.8%

For the entire treatment period, a trend toward reduced food intake (associated with reduced weight gain) at 125 and 225 mg base/kg/day was considered an adverse dose-related response to berusudil treatment. Trends in increased food intake and mean body weight gain after treatment at 125 and 225 mg base/kg/day were considered indicative of a rebound/recovery effect.

Maternal food intake is summarized in Table 16 below: Table 16. Maternal food intake / % difference from control Group ( mg base /kg/ day) 0 50 125 225 GD 6-10 159.5 149.4 ^{117.9b 61.2b} GD 6-19 156.4 142.7/-8.8% ^103.1b /-34.1% ^78.7b /-49.7% GD 19-29 131.8 136.0 ^{149.9a 155.3b} GD 0-29 145.7 141.1 ^128.8a /-11.6% ^119.8b /-17.8% ^a is significantly different from the control: p ＜ 0.05 ^b is significantly different from the control: p ＜ 0.01 Postmortem examination of uterus, ovaries and fetus

On GD 29, each surviving primary study female was euthanized by intravenous injection of sodium pentobarbital/euthanasia solution, followed by guaranteed death by SOP-approved methods, and immediate laparotomy hysterectomy. The skin is turned over through the ventral midline incision to examine the breast tissue and locate any subcutaneous mass. The abdominal cavity is then opened, and the uterus is exposed. The uterus was removed, and the gravid uterine weight was recorded. Starting from the distal end of the left uterine horn, the positions of viable and nonviable fetuses, early and late resorptions for each uterine horn, and the total number of implantations were recorded. The number of corpus luteum on each ovary was also recorded.

The fetus is removed by making a dorsal incision longitudinally along the two uterine horns. The embryonic membranes of each fetus are gently removed and each fetus is separated from the placenta, allowing the umbilical cord to be fully stretched. Examine the placenta roughly.

Appearing non-pregnant uteri from females were opened and placed in 10% ammonium sulfide solution to detect implantation sites. If no lesions were detected, the female was considered not pregnant.

When comparing in utero values to those in the control group, treatment-related adverse effects were not apparent at 50 mg base/kg/day.

Post-implantation loss rates (%/litter) at 125 and 225 mg base/kg/day were 7.42% and 17.72%, respectively, compared to 4.07%/litter in the control group. Dose-related increase in post-implantation loss rate (%/litter), corresponding increase in mean number absorbed (early and late combined), and decrease in mean number of viable fetuses/litter size at 125 and 225 mg base/kg/day Considered therapeutically relevant and undesirable.

At 225 mg base/kg/day, the mean fetal weight of male fetuses was reduced (statistically significant), resulting in a lower mean fetal weight of the combined sexes. Compared with the control value of 42.05 g, the male fetal weight value (litter incidence rate) was 38.92 g; the difference with the control group was -7.44%. Compared with the control value of 40.96 g, the average fetal weight of the combined sex at 225 mg base/kg/day was 38.60 g; the difference with the parallel control group was -5.76%. The average fetal weight for combined genders in historical data is 41.426 g. Effects on mean fetal weight at 225 mg base/kg/day were considered treatment-related and adverse.

In utero data are summarized in Table 17 below. The fetal weight information package is reported in Table 18 below. Table 17. Summary of uterine examinations Group ( mg base /kg/ day) 0 50 125 225 Number of litters evaluated by GD 29 twenty three twenty two 19 twenty one GD 29 has all absorbed quantities 0 0 0 2 Number of surviving fetuses in GD 29 litters twenty three twenty two 19 19 Average post-implantation loss rate (%/litter) 4.07 2.42 7.42 17.72 Average number absorbed (combination) 0.3 0.2 0.7 ^1.5a Average early absorption number 0.3 0.2 0.5 ^1.4a Average number of surviving littermates 8.4 8.5 8.0 7.4 aSignificantly different from control: ^p < 0.05 Table 18. Summary of mean fetal weight ( g ) / % difference from control Group ( mg base /kg/ day) 0 50 125 225 Male fetal weight 42.05 41.64 41.61 ^38.92a /-7.44% Female fetal weight 39.24 40.25 41.14 38.94 Combined gender fetal weight 40.96 40.97 41.09 38.60/-5.76% aSignificantly different from control: ^p < 0.05

Fetal abnormalities, short tail (global), occurred only at 225 mg base/kg/day and were considered an adverse treatment-related reaction. Five fetuses from two litters had this abnormality at 225 mg base/kg/day. The litter/fetus incidence rate was 10.5%/3.2%, compared with 5.0%/0.6% in historical data.

External fetal anomalies are summarized in Tables 19 and 20 below: Table 19. Fetal external malformations Group (mg base /kg/ day ) 0 50 125 225 Number of litters (number of fetuses) 1 (4.3) 1 (0.5) 1 (4.5) 1 (0.5) 0 (0.0) 0 (0.0) 2 (10.5) 5 (3.2) Table 20. Fetal external malformations: short tail Group (mg base /kg/ day ) 0 50 125 225 Number of litters (number of fetuses) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 2 (10.5) 5 (3.2)

Fetal variant tail curvature (overall) was seen at 225 mg base/kg/day in two fetuses from one litter, two of which, in addition to the caudal deformity seen on skeletal examination, also showed the above-mentioned short tail Abnormal. In this study, the litter (fetus) incidence of this variant was 5.3% (1.3%), and, respectively, the incidence in historical data was 4.3% (0.5%). The stated incidence is considered treatment-related and unfavorable given the association with brachycaudal anomalies.

The overall incidence of fetal visceral variants was increased at 125 and 225 mg base/kg/day compared with controls, however, there was no dose-related trend and they were overall limited in incidence and were consistent with historical data. Typical of those seen in. This included fetal variant ureteral malposition, which had a statistically significant increase (on a litter basis) at 125 (litter incidence 21.1%) and 225 mg base/kg/day (litter incidence 26.3%). However, in historical data, the incidence of ureteral malpositioning (fetuses) was 42.1% (7.7%), and therefore, this variant is not considered an adverse treatment-related effect.

Fetal visceral observations are summarized in Table 21, and fetal skeletal malformations are reported in Table 22. Table 21. Fetal visceral variations Group (mg base /kg/ day ) 0 50 125 225 Number of litters (number of fetuses) 6 (26.1) 6 (3.1) 4 (18.2) 6 (3.2) 7 (36.8) 11 (7.2) 8 (42.1) 12 (7.7) Table 22. Fetal skeletal malformations Group (mg base /kg/ day ) 0 50 125 225 Number of litters (number of fetuses) 3 (13.0) 4 (2.1) 6 (27.3) 8 (4.3) 6 (31.6) 10 (6.6) 10 (52.6) 19 (12.2)

The overall incidence of fetal skeletal malformations increased at 50, 125 and 225 mg base/kg/day, however there was no clear dose-related trend at 50 and 125 mg base/kg/day. However, there was a trend in the fetal skeletal data at 225 mg base/kg/day whereby the overall incidence of fetal skeletal malformations increased and specific skeletal malformations increased, compared with concurrent controls, which were typically not only increases, and are outside the range seen in historical comparisons. Fetal skeletal findings that primarily contributed to the overall increase in abnormalities at 225 mg base/kg/day consisted of thoracic and thoracic spinal deformities, including the following findings: one or more rib branches (statistically significant), fusion or malformation, a or fusion of multiple sternal segments as well as neural arch fusion, malpositioning, and shape deformity (statistically significant). The finding of one or more rib branches occurred in 21.2% (2.6%) of the litters/fetus; in historical data the incidence was 5.0% (0.5%). The finding of fusion of one or more ribs occurred in 15.8% (2.6%) of the litters/fetus; the historical rate was 14.3% (1.7%). Deformity of one or more ribs was found in 15.8% (1.9%) of the litters/fetus; the historical rate was 5.9% (0.7%). The finding of fusion of one or more sternal segments occurred with a litter/fetal incidence rate of 31.6% (4.5%); the historical incidence rate was 26.1% (3.9%).

Neural arch (thoracic) fusion was found to occur in 10.5% (1.9%) of the litters/fetus; the rate in historical data was 9.5% (1.1%). Neural arch (thoracic) malpositioning was found in 10.5% (1.3%) of the litters/fetus; the historical rate was 5.3% (0.6%). Neural arch (thoracic cavity) shape deformities were found in 21.1% (2.6%) of the litters/fetus; the historical rate was 9.5% (1.1%). These skeletal deformities observed at 225 mg base/kg/day are considered begrusudil-related and undesirable.

Additionally, two fetal skeletal variations were increased (statistically significant) at 225 mg base/kg/day and are considered potentially adverse and treatment-related. One of these findings was seen in one or more cervical vertebrae, classified as central or semi-central, which occurred in 21.1% (2.6%) of the litters/fetus; historical data showed an incidence of 5.3% (0.6%) . Another fetal skeletal variation that increased at 225 mg base/kg/day was the finding of one or more additional sternal segments, which occurred at a litter/fetal incidence of 21.1% (4.5%); the historical incidence was 5.3 % (1.5%). Macroscopic observation of mother body

During planned necropsy examinations on GD 29, maternal necropsy findings from the three animals that did not survive revealed the following. Two animals aborted on GD 19, respectively at 125 and 225 mg base/kg/day (animal numbers 251 and 282, respectively). One subject (dosed at 125 mg base/kg/day) had mild/minimal amounts of red fluid in the bladder and uterus, and another animal dosed at 225 mg base/kg/day was found to have increased GD 9 deaths. The cause of death was not determined at necropsy; the animal had yellow discoloration of the adipose tissue (moderate, icteric) and multiple red lesions on the thymus (mild). Death was considered an adverse treatment-related effect. Toxicokinetic analysis

Exposure to berusudil, KD025m1, and KD025m2 increased as berusudil dose levels increased from 50 mg base/kg to 225 mg base/kg. The increase in mean C _max values of berusudil, KD025m1, and KD025m2 in pregnant rabbits was generally dose-proportional at GD 6 and GD 18, and the increase in AUC _0-24 was greater than dose-proportional at GD 6 and GD 18; however, at GD 6 and GD 18 AUC _0-24 was highly variable at the 225 mg base/kg dose level. The AUC _0-24 values at GD18 were generally similar or lower than the AUC _0-24 values at GD 6, indicating that no observed berusudil, Significant accumulation of KD025m1 and KD025m2.

Mean AUC _0-24 metabolite to maternal ratios indicate that berusuudil is converted to KD025m1 and KD025m2 in pregnant rabbits following oral gavage administration. For KD025m1 and KD025m2, the metabolite to parent ratio ranged from 0.344 to 0.820 and 0.652 to 1.98, respectively. The results of the TK analysis are reported in Table 23 below. Table 23. Summary of mean berusudil, KD025m1 , KD025m2 _Cmax and AUC _0-24 in plasma of pregnant rabbits Gap (GD) dose group Dosage level (mg base /kg) Berushudil KD025m1 KD025m2 C _max (ng/mL) AUC _0-24 (ng*hr/mL) C _max (ng/mL) AUC _0-24 (ng*hr/mL) C _max (ng/mL) AUC _0-24 (ng*hr/mL) 6 6 50 919 3870 750 2470 1170 4180 7 125 3030 21400 1250 7490 2180 13600 8 225 4140 55700 1790 21300 3190 42400 18 6 50 437 1590 569 1220 1120 2470 7 125 1670 9290 1350 5740 3610 16700 8 225 2580 23500 1680 14800 4620 41500 result

When berusudil was administered to pregnant rabbits at 50, 125, and 225 mg base/kg/day from gestation days 6 to 18, maternal and developmental toxicity occurred at 125 and 225 mg base/kg/day. . One animal was found to miscarry each at 125 and 225 mg base/kg/day, and one animal died at 225 mg base/kg/day.

Effects on body weight and food intake during the treatment periods at 125 and 225 mg base/kg/day were considered adverse and related to berusudil treatment. Dose-related increases in post-implantation losses (consisting of early and late absorption) and reductions in viable fetuses/litter sizes at 125 and 225 mg base/kg/day are considered treatment-related and undesirable. Effects on fetal weight occurred only at 225 mg base/kg/day, and fetal examination data (external and skeletal) revealed effects on fetal development only at 225 mg base/kg/day. In summary, a dose level of 50 mg base/kg/day is considered the NOAEL for maternal and developmental toxicity.

For the parent compound berusudil, the 50 mg base/kg/day dose level corresponds to a parent Cmax of 437 ng/mL and a parent AUC0-24 of 1590 ng·hr at the end of the dosing regimen (GD 18) /mL; for KD025m1, the parent Cmax was 569 ng/mL and the AUC0-24 was 1220 ng·hr/mL; and for KD025m2, the parent Cmax was 1120 ng/mL and the AUC0-24 was 2470 ng·hr/mL. Example 5 : Combination Study of Fertility and Early Embryonic Development to Implantation in Sprague - Dürer Rats Study Objectives and Design

This study was conducted to determine the effects of berusuudil on the female estrous cycle, fallopian tube transit, embryo implantation and development, and to examine functional effects on male fertility. The study design used treatment-naïve animals of both sexes, combined treatments for both sexes, and provided a recovery period for treated males based on the study results. The study also included toxicokinetic evaluations to determine exposure/toxicity relationships.

Male and female Sprague-Dürer (SD) rats (approximately 7 to 10 weeks old) were obtained from Charles River Laboratories, Raleigh, NC. Using a standard weight-based randomization procedure, 273 male and 273 female animals (randomized weighing 229 g to 497 g and 169 g to 242 g, respectively) were assigned to control, treatment, and toxicokinetic ( TK) group. The design of the study is shown in Table 24. The dose volume for each group is 10 mL/kg, providing dose concentrations in mg/base/mL at one-tenth the dose level). (In Table 24, T = treated; U = untreated). Table 24 : Combined Fertility and Early Embryonic Development Study Design group Dosage level ( mg base /kg/ day) Number of animals main research restore toxicokinetics Male ( T ) Female ( T ) Female ( U ) Male ( T ) Female ( U ) Male ( T ) Female ( T ) 1 0 ^25a 2 50 ^25a 3 150 ^25a 4 275 ^25a 5 0 ^25b 25 6 50 ^25b 25 7 150 ^25b 25 8 275 ^25b 25 9 0 ^{10c 10c} 10 50 ^{10c 10c} 11 150 ^{10c 10c} 12 275 13 0 ^{10c 10c} 3d 3e 14 50 10 ^{days 10e} 15 150 10 ^{days 10e} 16 275 10 ^days 10 ^e Total number of animals: 100 100 100 40 40 33 33 Treat females 14 days before pairing with untreated males; males remain naïve until the end of the first mating period; schedule males to arrive at 7 weeks of age and allow a minimum of one week to acclimate. b Males were dosed starting after pairing with a treated female (first mating period) and continued for 70 days before pairing with an untreated female (second mating period) until GD13 uterine examination. c Males assigned to recovery were dosed for 70 days and allowed to recover for 70 days before being paired (1:1) with untreated females; pairing with unmedicated females was based on study results. d Three TK control males from 1 group were blood collected at one time point on day 1 and day 70 of the study; 5 TK males from 2 groups were collected at alternating times on day 1 of the study (6 time points) and Blood was collected at alternating times (7 time points) on day 70; 3 TK control females in 1 cohort were bled at one time point on study days 1 and 14; 5 TK females in 2 cohorts were Blood collection at alternating time on day 1 (6 time points) and alternating time on day 14 (7 time points)

All animals were given a detailed clinical examination before selection, and body weights were recorded upon receipt and before selection. Additionally, males assigned to groups 5-8 were given detailed clinical observations weekly and body weights were recorded weekly during the acclimatization period.

Animals were housed individually in solid-bottomed cages with non-aromatic bedding in an environmentally controlled room except during pairing periods. During the pairing period, rats (one male and one female from corresponding groups) were cohoused in the male's cage. During the acclimatization period, animals were observed twice daily for general health and any signs of disease. invest

Dosage levels of 0, 50, 150, and 275 mg base/kg/day and a dose volume of 10 mL/kg were administered via oral gavage in the morning at approximately the same time each day (±2 hours from Day 1 dosing). All treated animals were administered vehicle and berusudil once daily. The high dose level (275 mg base/kg/day) provides the opportunity to have some mild toxicity and allows evaluation of fertility and early embryonic development toxicity at exposure levels approximately 4 times higher than the expected highest steady-state clinical exposure. The low (50 mg base/kg/day) and mid-dose levels (150 mg base/kg/day) were selected to demonstrate dose-dependent responses and are expected to be clinically relevant (maximum expected steady-state clinical exposure, respectively). Approximately 0.5 times to 1 times and 2 times the amount).

Dosing was initiated at 11 weeks of age and 70 days before pairing (with untreated females) for treated males, and 14 days before pairing (with untreated males) for treated females. Dosing of males continued through the mating period and post-mating period until euthanasia, while dosing of females continued through the mating period until GD 7. After the mating period is completed, females showing no signs of mating are dosed for 7 days. Males designated for recovery were dosed for 70 days and then received a 77-day recovery period.

Additionally, TK animals received vehicle or berusudil in the same manner and at the same dose levels and volumes as the main study group. Male and female TK animals were dosed for 70 and 14 days respectively. Dosing of TK males and females was started simultaneously and continued until euthanasia. Prior to dosing, bring frozen daily aliquots of the dosing formulation to room temperature with continuous stirring for at least 30 minutes. Stir the vehicle and berusudil formulation continuously prior to and throughout dosing. Individual dosage is based on recent body weight. Observe and analyze

Animals were observed cageside for detailed clinical changes, body weight, food intake, estrous cycle determination, plasma analysis, sperm analysis, toxicokinetic analysis and anatomical pathology, including uterine and ovarian examination.

Cage-side observations were conducted at least twice daily. Observe all animals for morbidity, mortality, injuries, and availability of food and water.

Detailed clinical observations were made during the treatment phase: for males, twice a week during the treatment period (4 hours +/- 1 hour on the dosing day), and for females, daily during the treatment period (4 hours +/- 1 hour on the dosing day). 1 hour). Detailed clinical observations were performed weekly during the non-treatment phase (males and females) and also on GD 13 before regular uterine examinations (females). Such observations include, but are not limited to, evaluation of the skin, fur, eyes, ears, nose, mouth, chest, abdomen, external genitalia, extremities and feet, and evaluation of breathing.

For each endpoint, compare the treatment group to the control group using the analysis listed in Table 25. As shown, some endpoint data are transformed by arcsine square root transformation before performing the specified analysis. Table 25. Statistical analysis end point type of analysis Maternal life data Body weight: pre-mating, gestation, post-mating (males) Weight changes: between each weighing interval, throughout the pre-mating period (males and females), gestation, post-mating (males) Food intake: Pre-mating, pregnancy, post-mating (male) Pairwise comparisons of groups Pairwise comparisons of groups Pairwise comparisons of groups Fertility index Mating interval Fertility index (male and female) Mating index (male and female) Fertility index (male and female) Estrus cycle (average cycle time and number of cycles/periods) pairwise comparison of groups Fisher's exact test Fisher's exact test Fisher's exact test pairwise comparison of groups Pathology Male reproductive organ weight (absolute weight and relative to body weight) Female reproductive organ weight (absolute weight) Sperm analysis (abnormality % and motility %) Sperm analysis (concentration) pairwise comparison of groups pairwise comparison of groups arcsine square root transform pairwise comparison of groups Total number of corpus luteum in uterine examination /total number of implantations in female rats/viable fetuses in female rats/number of absorbed females/pre-implantation loss in female rats % post-implantation loss Make up a pair comparison Make up a pair comparison Make up a pair comparison Make up a pair comparison Arc sine square root transform Arc sine square root transform Toxicokinetic analysis

A total of 402 berusudil samples, 402 KD025m1 samples, and 402 KD025m2 samples were analyzed using protein precipitation, followed by high-performance liquid chromatography followed by tandem mass spectrometry detection (LC-MS/MS). Berusudil exposure increased as dose levels increased from 50 mg base/kg/day to 275 mg base/kg/day.

Increases in _Cmax and AUC _0-24 values were generally less than dose proportional for females on day 1 and approximately dose proportional for males on day 1, and at day 14 and day 70 for females and males, respectively. Roughly proportional to dose. Gender differences in berusudil C _max and AUC _0-24 values were less than 2-fold, with the following exceptions: Day 1 Group 14 (50 mg base/kg/day), where the corresponding values for females were approximately 2.10 higher than those for males, respectively times and 2.44 times. Cumulative ratios of AUC _0-24 ranged from 0.533 to 1.72 at day 14 for females and from 0.859 to 1.98 at day 70 for males. The information is summarized in Table 26. Table 26. Summary of C _max , AUC _0-24 and AR of berusudil in rat plasma Interval (days) dose group Dosage level (mg base/kg) gender C _max (ng/mL) AUC _0-24 (hg hr/mL) AR AUC _0-24 1 14 50 M 2170 20100 NA F 4540 49200 NA 15 150 M 5370 81600 NA F 5670 95700 NA 16 275 M 7200 96400 NA F 7050 122000 NA 14 14 50 F 3200 26200 0.533 15 150 F 9860 99500 1.04 16 275 F 14900 209000 1.72 70 14 50 M 2480 21600 1.07 15 150 M 10100 70100 0.859 16 275 M 20400 191000 1.98 AR - Cumulative Ratio NA - not applicable M - Male F - Female

Exposure to KD025m1 increased as berusudil dose levels were increased from 50 to 275 mg base/kg/day. C _max and AUC _0-24 values in males increased less than dose proportionally at dose levels between 50 and 150 mg base/kg/day and at dose levels between 150 and 275 mg base/kg/day The dose is roughly proportional to the dose.

Female C _max and AUC _0-24 values increased approximately proportionally to dose. Sex differences in KD025m1 C _max and AUC _0-24 values were less than 2-fold, with the following exceptions: Group 14 (50 mg base/kg/day), where the corresponding values for males were approximately 2.10 to 2.62 times higher than for females. Cumulative ratios of AUC _0-24 ranged from 0.738 to 1.49 at day 14 for females and from 0.634 to 1.24 at day 70 for males. For AUC _0-24 , metabolite to parent ratios ranged from 0.102 to 0.520. The information is summarized in Table 27. Table 27. Summary of KD025m1 _Cmax , AUC _0-24 and AR in rat plasma Interval (days) dose group Dosage level (mg base/kg) gender C _max (ng/mL) AUC _0-24 (hg hr/mL) AR AUC _0-24 1 14 50 M 934 10500 NA F 369 5000 NA 15 150 M 1420 25600 NA F 1050 16100 NA 16 275 M 2820 35600 NA F 2020 24900 NA 14 14 50 F 294 3690 0.738 15 150 F 1260 18200 1.13 16 275 F 2290 37000 1.49 70 14 50 M 770 8460 0.807 16 150 M 1800 16200 0.634 16 275 M 3350 44200 1.24 AR - Cumulative Ratio NA - not applicable M - male F - Female

Exposure to KD025m2 increased as the berusudil dose level increased from 50 to 275 mg base/kg/day. C _max and AUC _0-24 values in males increased less than dose proportionally at dose levels between 50 and 150 mg base/kg/day and at dose levels between 150 and 275 mg base/kg/day The dose is roughly proportional to the dose.

Increases in _Cmax and AUC _0-24 values for females were approximately dose-proportional on day 1 and were greater than dose-proportional on day 14. Gender differences in KD025m2 C _max and AUC _0-24 values were generally less than 2-fold, with the following exceptions: Group 14 (50 mg base/kg/day), where the corresponding values for males were approximately 2.58 to 3.70 times higher than for females. No significant accumulation (<2-fold) of KD025m2 was observed after multiple administrations of berusuudil in rats. Cumulative ratios of AUC _0-24 ranged from 0.657 to 1.80 at day 14 for females and from 0.701 to 1.35 at day 70 for males. For AUC _0-24 , metabolite to parent ratios ranged from 0.0429 to 0.270. The information is summarized in Table 28. Table 28. Summary of KD025m2 _Cmax , AUC _0-24 and AR in rat plasma Interval (days) dose group Dosage level (mg base/kg) gender C _max (ng/mL) AUC _0-24 (hg hr/mL) AR AUC _0-24 1 14 50 M 501 5440 NA F 135 2110 NA 15 150 M 686 13600 NA F 398 6860 NA 16 275 M 1680 21500 NA F 925 11500 NA 14 14 50 F 125 1390 0.657 15 150 F 523 7110 1.04 16 275 F 1480 20700 1.80 70 14 50 M 410 4080 0.750 15 150 M 1070 9520 0.701 16 275 M 1900 28900 1.35 AR - Cumulative Ratio NA - not applicable M - Male F - Female

In this fertility and early embryonic development toxicity study with berusudil, effects were observed in treated males and females at 150 and 275 mg base/kg/day.

Adverse clinical observations of abnormal feces (little/no or discoloration) and emaciated body condition were observed in treated females at 275 mg base/kg/day.

Additionally, females at 150 and 275 mg base/kg/day had lower mean body weight, mean body weight change, and reduced mean food intake throughout the treatment period and were considered to be berusudil-related and undesirable. of. Lower mean body weight and/or body weight variation and reduced food intake were also observed in females at 50 mg base/kg/day. These differences at 50 mg base/kg/day, although possibly related to berusuudil, were in fact incidental, minor, and not considered adverse.

At 275 mg base/kg/day, berusudil-related increases in mean post-implantation loss and mean number of absorbed embryos were observed in treated females and were associated with lower mean number of viable embryos. Ovarian and uterine parameters (corpus luteum count, implantation sites, number of viable embryos and resorptions, and pre- and post-implantation losses) were not affected by berusudil treatment at 50 and 150 mg base/kg/day . Reproductive and fertility indices of treated females were unaffected at all dose levels evaluated. No begrusudil-related macroscopic findings or organ weight differences were observed in treated females.

Adverse clinical observations of abnormal feces (little/none) and emaciated body condition were observed in treated males at 275 mg base/kg/day. In males, salivation was observed at 275 and 150 mg base/kg/day, and while potentially related to berusudil, this finding was not dose-responsive and was in fact sporadic and therefore not considered adverse. . Additionally, males at 150 and 275 mg base/kg/day had lower mean body weight, mean body weight change, and reduced mean food intake throughout the treatment period and were considered to be berusudil-related and undesirable. of.

During the recovery period, the frequency of most of these findings decreased at 150 and 275 mg base/kg/day, indicating reversibility of toxicity. Lower mean body weight and/or body weight variation and reduced food intake were also observed in males at 50 mg base/kg/day. These differences at 50 mg base/kg/day, although possibly related to berusuudil, were in fact incidental, minor, and not considered adverse.

At 275 mg base/kg/day, fertility and fecundity indexes in treated males were low (72% and 75%, respectively) and were considered to be berusudil-related and undesirable. During the recovery phase, no effects were seen on reproduction and fertility indices (mating, fertility and fecundity parameters) at 275 mg base/kg/day. Reproductive and fertility indices were not affected by berusudil treatment at 50 and 150 mg base/kg/day.

Average number of implantation sites and viable embryos among 6 non-pregnant untreated females (25 untreated females mated with treated males) at 275 mg base/kg/day The average quantity decreased. These differences in pregnancy outcomes and uterine parameters at 275 mg base/kg/day are thought to be related to the abnormal sperm evaluation (low motility, low mean sperm count, and increased percentage of abnormal sperm) observed in treated males, and Considered to be related and undesirable to begrusudil. In males treated with 275 mg/kg/day, reproduction, fertility and sperm parameters were not affected during the recovery period. Male reproduction and fertility parameters were unaffected at 50 and 150 mg base/kg/day.

At 275 mg/kg/dose, berusudil-related macroscopic observations of small testicles (left and right) and epididymis (left, right, and right tail) were observed at terminal and recovery necropsy and were associated with reduced Mean organ weight (absolute) correlation. Additionally, at 275 mg/kg/day, the testes (minimal to severe degeneration/atrophy) and epididymis (minimal to mild luminal cell fragments) were noted in approximately 64% and 68% of terminal males, respectively; and upon return to necropsy , the percentages of treated males in which these findings were observed were approximately 30% and 40%, respectively. Macroscopic findings, organ weights, and microscopic findings were not affected by treatment with 50 and 150 mg base/kg/day.

Exposure C _max and AUC _0-24 values as assessed by berusudil and its metabolites KD025m1 and KD025m2 increased as berusudil dose levels increased from 50 to 275 mg base/kg/day . Increases in berusudil C _max and AUC _0-24 values were generally less than dose-proportional for females on day 1 and approximately dose-proportional for males on day 1 and on day 14 for both males and females and day 70 are roughly proportional to dose.

The increase in C _max and AUC _0-24 values for KD025m1 and KD025m2 in males was less than dose proportional at dose levels between 50 mg and 150 mg base/kg/day, and at 150 mg base/kg/day to 275 mg Base/kg/day dose levels are approximately proportional to dose. Increases in C _max and AUC _0-24 values for KD025m1 and KD025m2 in females were approximately dose-proportional, with the following exceptions: KD025m2 on day 14, where the increases were greater than dose proportional.

Gender differences in berusudil C _max and AUC _0-24 values were less than 2-fold, with the following exceptions: Day 1 Group 14 (50 mg base/kg/day), where the corresponding values for females were approximately 2.10 times higher than those for males, respectively times and 2.44 times.

Sex differences in C _max and AUC _0-24 values for KD025m1 and KD025m2 were generally less than 2-fold, with the following exceptions: Group 14 (50 mg base/kg/day), where the corresponding values for males were approximately 2.10 to 3.70 times higher than for females.

For AUC _0-24 of KD025m1, the metabolite to parent ratio ranged from 0.102 to 0.520. For the AUC _0-24 of KD025m2, the metabolite to parent ratio ranged from 0.0429 to 0.270.

Based on these results, the NOAEL for the general toxicity endpoint is considered to be 50 mg base/kg/day for male and female rats [AUC _0-24 is 21600 ng∙hr/mL (males) and 26200 ng∙hr/mL (female) and C _max is 2480 ng/mL (male) and 3200 ng/mL (female)].

For male and female reproductive performance and fertility, the NOAEL was considered to be 150 mg base/kg/day and 275 mg base/kg/day, respectively [AUC _0-24 of 70100 ng∙hr/mL and C _max of 10100 ng/mL (males) and AUC _0-24 of 209000 ng∙hr/mL and C _max of 14900 ng/mL (females)].

In treated females, the NOAEL for ovarian and uterine parameters was considered to be 150 mg base/kg/day (AUC _0-24 of 99500 ng∙hr/mL and C _max of 9860 ng/mL). Example 6 : US REZUROCK™ (Berusudil) FDA label

----------------------------Indications and Usage------------------ -------

REZUROCK is a kinase inhibitor indicated for the treatment of adult and pediatric patients 12 years of age and older with chronic graft-versus-host disease (chronic GVHD) after failure of at least two prior lines of systemic therapy. (1)

-----------------------Dosage and Administration----------------------

Recommended dose: 200 mg taken orally once daily with food. (2.1)

--------------------------Dosage Forms and Specifications--------------------------

Tablet: 200 mg. (3)

----------------------------------Contraindications------------------ ----------

without. (4)

-----------------------Warnings and Precautions----------------------

Embryo-Fetal Toxicity: May cause harm to the fetus. Inform females of reproductive potential of the potential risks to the fetus and to use effective contraception. (5.1, 8.1, 8.3)

----------------------------------Drug Interactions---------------- -------------

Strong CYP3A Inducers: Increase REZUROCK dose to 200 mg twice daily. (7.1)

Proton Pump Inhibitors: Increase REZUROCK dose to 200 mg twice daily. (7.1)

----------------------------------Adverse reactions------------------ ----------

The most common (≥ 20%) adverse reactions (including laboratory abnormalities) are infection, asthenia, nausea, diarrhea, dyspnea, cough, edema, bleeding, abdominal pain, musculoskeletal pain, headache, decreased phosphate, gamma glutamine Increased transferases, lymphopenia, and hypertension. (6.1)

-----------------------Medicine for special groups-----------------------

Lactation period: Breastfeeding is not recommended. (8.2)

See 17 for patient counseling information and FDA-approved patient labeling. Full Prescribing Information1Indications and Uses

REZUROCK is indicated for the treatment of adult and pediatric patients 12 years of age and older with chronic graft-versus-host disease (chronic GVHD) after failure of at least two prior lines of systemic therapy. 2 Doses and Administering 2.1 Recommended Doses

The recommended dose of REZUROCK is 200 mg administered orally once daily until progression of chronic GVHD requires new systemic therapy.

Instruct patients as follows: • Swallow REZUROCK lozenges whole. Do not cut, crush, or chew lozenges. • Take REZUROCK at approximately the same time each day with a meal [ see Clinical Pharmacology (12.3)] . • If a dose of REZUROCK is missed, instruct the patient not to take additional doses to make up for the missed dose.

Treatment with REZUROCK has not been studied in patients with pre-existing severe renal or hepatic impairment. In patients with pre-existing severe renal or hepatic impairment, consider the risks and potential benefits before initiating treatment with REZUROCK [ see Clinical Pharmacology (12.3)] . 2.2 Dose modification of adverse reactions

Monitor total bilirubin, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) at least monthly. Modify REZUROCK dosage for adverse reactions according to Table 29 .

Table 29: REZUROCK Recommended Dose Modifications for Adverse Reactions adverse reactions Severity * REZUROCK dosage modification Hepatotoxicity [ see Adverse Reactions (6.1)] Grade 3 AST or ALT (5x to 20x ULN) or Grade 2 bilirubin (1.5x to 3x ULN) Maintain REZUROCK until bilirubin, AST, and ALT return to Grade 0-1, then resume REZUROCK at the recommended dose. Grade 4 AST or ALT (more than 20x ULN) or ≥ Grade 3 bilirubin (more than 3x ULN) Permanently abort REZUROCK. Other adverse reactions [ see Adverse Reactions Level 3 Maintain REZUROCK until recovery to level 0-1, then resume REZUROCK at recommended dosage levels. (6.1)] Level 4 Permanently abort REZUROCK. *Based on CTCAE v 4.03 2.3 Dosage modification due to drug interactions

Strong CYP3A inducer

When coadministered with strong CYP3A inducers, increase the dose of REZUROCK to 200 mg twice daily [ see Drug Interactions (7.1)] .

proton pump inhibitor

When coadministered with a proton pump inhibitor, increase the dose of REZUROCK to 200 mg twice daily [ see Drug Interactions (7.1)] . 3 dosage forms and specifications

Each 200 mg tablet is a pale yellow film-coated oval-shaped tablet with a debossed pattern of "KDM" on one side and a debossed pattern of "200" on the other side. 4 Contraindications

without. 5 Warnings and Precautions 5.1 Embryo - fetal toxicity

Based on findings in animals and its mechanism of action, REZUROCK may cause fetal harm when administered to pregnant women. In animal reproduction studies, administration of berusudil to pregnant rats and rabbits during organogenesis produced adverse developmental outcomes, including embryo-fetal mortality and malformation ratio at maternal exposure (AUC) at recommended doses. Sometimes low in patients. Inform pregnant women of potential risks to the fetus. Advise females of reproductive potential and males of reproductive potential who have female partners to use effective contraception during treatment with REZUROCK and for at least one week after the last dose [ see Medication in Specific Populations (8.1 , 8.3) , Nonclinical Toxicology (13.1)] . 6 Adverse reactions 6.1 Clinical trial experience

Because clinical trials are conducted under widely varying conditions, adverse reaction rates observed in clinical trials of one drug cannot be directly compared to adverse reaction rates in clinical trials of another drug and it may not be reflected in practice. Adverse reaction rates observed.

chronic graft versus host disease

In two clinical trials (Study KD025-213 and Study KD025-208), 83 adult patients with chronic GVHD were treated with REZUROCK 200 mg once daily [ see Clinical Studies (14.1)] . The median duration of treatment was 9.2 months (range, 0.5 to 44.7 months).

One patient reported fatal adverse reactions, including severe nausea, vomiting, diarrhea, and multiorgan failure.

Permanent discontinuation of REZUROCK due to adverse reactions occurred in 18% of patients. Adverse reactions leading to permanent discontinuation of REZUROCK included nausea (4%) in >3% of patients. Adverse reactions leading to dose interruption occurred in 29% of patients. Adverse reactions leading to dose discontinuation in ≥2% were infection (11%), diarrhea (4%) and asthenia, dyspnea, bleeding, hypotension, abnormal liver function tests, nausea, pyrexia, edema, and renal failure (2 each) %).

The most common (≥ 20%) adverse reactions (including laboratory abnormalities) are infection, asthenia, nausea, diarrhea, dyspnea, cough, edema, bleeding, abdominal pain, musculoskeletal pain, headache, decreased phosphate, gamma glutamine Increased transferases, lymphopenia, and hypertension.

Table 30 summarizes non-laboratory adverse reactions.

Table 30: Non-laboratory adverse reactions in > 10% of patients with chronic GVHD treated with REZUROCK adverse reactions REZUROCK 200 mg once daily (N = 83) All ratings Level 3-4 ( % ) Infections and infestations Infection (pathogen not specified) ^a 53 16 Viral ^infectionb 19 4 Bacterial ^infectionc 16 4 Systemic disorders and administration site conditions ^Weak 46 4 ^Edemae 27 1 Fever 18 1 gastrointestinal tract Disgusting ^f 42 4 Diarrhea 35 5 Abdominal ^paing twenty two 1 difficulty swallowing 16 0 Respiratory system, chest and mediastinum ^Difficulty breathingh 33 5 ^coughi 30 0 nasal congestion 12 0 Blood vessel Bleeding ^j twenty three 5 high blood pressure twenty one 7 Musculoskeletal and connective tissue Musculoskeletal ^paink twenty two 4 adverse reactions REZUROCK 200 mg once daily (N = 83) All ratings Level 3-4 ( % ) muscle spasms 17 0 joint pain 15 2 nervous system headache ^l twenty one 0 metabolism and nutrition decreased appetite 17 1 skin and subcutaneous ^rashm 12 0 itching ⁿ 11 0 a Infections with unspecified pathogens, including acute sinusitis, device-related infections, ear infections, folliculitis, gastroenteritis, gastrointestinal infections, hordeolumitis, infectious colitis, pulmonary infections, skin infections, dental infections, urinary tract infections Tract infection, wound infection, upper respiratory tract infection, pneumonia, conjunctivitis, sinusitis, respiratory tract infection, bronchitis, sepsis, septic shock. b Including influenza, rhinovirus infection, gastroenteritis virus, viral upper respiratory tract infection, bronchitis virus, EB viremia, EB virus infection, parainfluenza virus infection, varicella zoster virus infection, and viral infection. c Includes cellulitis, Helicobacter infection, staphylococcal bacteremia, catheter site cellulitis, Clostridium difficile colitis, Escherichia genus urinary tract infection, Escherichia coli gastroenteritis, Pseudomonas infection, urinary tract infection Infection with bacteria. d includes fatigue, weakness, and fatigue. e Including peripheral edema, generalized edema, facial edema, local edema, and edema. f Including nausea and vomiting. g Including abdominal pain, upper abdominal pain, and lower abdominal pain. h Including dyspnea, exertional dyspnea, apnea, orthopnea, sleep apnea syndrome. i Including cough and phlegm cough. jIncludes contusions, hematomas, epistaxis, increased tendency to bruise, conjunctival hemorrhage, bloody stools, oral bleeding, catheter site bleeding, hematuria, hemothorax, and purpura. k Including limb pain, back pain, flank pain, limb discomfort, musculoskeletal chest pain, neck pain, and musculoskeletal pain. l Including headaches and migraines. m Including rash, maculopapular rash, erythematous rash, generalized rash, exfoliative dermatitis. n Including itching and itching all over the body.

Table 31 summarizes laboratory abnormalities for REZUROCK.

Table 31 : Selected Laboratory Abnormalities in Chronic GVHD Patients Treated with REZUROCK REZUROCK 200 mg once daily Level 0-1 Baseline Maximum after using level 2-4 agent Maximum after using level 3-4 agent parameters ( N ) ( % ) ( % ) Chemistry Phosphate reduction 76 28 7 Increased gamma glutamine transferase 47 twenty one 11 Decreased calcium 82 12 1 Increased alkaline phosphatase 80 9 0 Increased potassium 82 7 1 Increased alanine aminotransferase 83 7 2 Increased creatinine 83 4 0 Hematology lymphopenia 62 29 13 Decreased hemoglobin 79 11 1 Thrombocytopenia 82 10 5 Decreased neutrophil count 83 8 4 7 Drug Interactions 7.1 Effects of Other Drugs on REZUROCK

Strong CYP3A inducer

Coadministration of REZUROCK with strong CYP3A inducers results in decreased berusudil exposure [ see Clinical Pharmacology (12.3)] , which may reduce the efficacy of REZUROCK. Increase the dose of REZUROCK when coadministered with strong CYP3A inducers [ see Dosage and Administration (2.3)] .

proton pump inhibitor

Coadministration of REZUROCK with proton pump inhibitors decreases berusudil exposure [ see Clinical Pharmacology (12.3)] , which may reduce the efficacy of REZUROCK. Increase the dose of REZUROCK when coadministered with a proton pump inhibitor [ see Dosage and Administration (2.3)] . 8 Medication for Special Groups 8.1 Pregnancy

Risk overview

Based on findings from animal studies and its mechanism of action [ see Clinical Pharmacology (12.1)] , REZUROCK may cause fetal harm when administered to pregnant women. There are no human data available on the use of REZUROCK in pregnant women to evaluate drug-related risks. In animal reproduction studies, administration of berusudil to pregnant rats and rabbits during organogenesis produced adverse developmental outcomes, including altered growth at maternal exposure (AUC), embryo-fetal mortality, and embryo- Fetal malformations are approximately ≥ 3 times (rat) and ≥ 0.07 times (rabbit) the human exposure (AUC) at recommended doses (see Animal Data). Inform pregnant women and women of reproductive potential of the potential risks to the fetus.

In the general U.S. population, the estimated background risks of major birth defects and miscarriage in clinically recognized pregnancies are 2% to 4% and 15% to 20%, respectively.

data

animal data

Conduct embryo-fetal development studies in rats in which berusudil was administered to pregnant animals during organogenesis in a pilot study at oral doses of 25, 50, 150, and 300 mg/kg/day, and Berusudil was administered to pregnant animals in pivotal studies at doses of 15, 50, and 150 mg/kg/day. In pilot studies, maternal toxicity and embryo-fetal developmental effects were observed. Maternal toxicity (decreased weight gain) occurred at doses of 150 and 300 mg/kg/day. Increased post-implantation losses occurred at 50 and 300 mg/kg/day. Fetal malformations including absence of anus and tail, omphalocele, and dome-shaped head were observed at ≥50 mg/kg/day. Rat exposure (AUC) at 50 mg/kg/day was approximately 3 times greater than human exposure at the recommended dose of 200 mg.

In an embryo-fetal development study in rabbits, administration of oral doses of berusudil to pregnant animals at 50, 125, and 225 mg/kg/day during organogenesis resulted in maternal toxicity and embryo-fetal developmental effects. Maternal toxicity (weight loss and death) was observed at doses ≥ 125 mg/kg/day. Embryo-fetal effects, including spontaneous abortion, increased post-implantation loss, decreased percentage of viable fetuses, malformations, and reduced fetal weight, were observed at doses ≥ 50 mg/kg/day. Deformities include those of the tail (shortness), ribs (branching, fusion, or deformation), sternum (fusion), and neural arches (fusion, malpositioning, and deformation). The rabbit exposure (AUC) at 50 mg/kg/day was approximately 0.07 times the human exposure at the recommended dose of 200 mg. 8.2 Lactation period

Risk overview

There are no data on the presence of berusudil or its metabolites in human milk or on the effects on the breastfed child or on milk production. Because berusudil may cause serious adverse reactions in breastfed children, advise nursing women not to breastfeed during treatment with REZUROCK and for at least one week after the last dose.

Women and men of reproductive potential

REZUROCK may cause fetal harm when administered to pregnant women [ see Medication in Specific Populations (8.1)] .

pregnancy test

Verify pregnancy status in females of reproductive potential before initiating treatment with REZUROCK. contraception

female

Advise females of reproductive potential to use effective contraception during treatment with REZUROCK and for at least one week after the last dose of REZUROCK. If this drug is used during pregnancy or if the patient becomes pregnant while taking this drug, the patient should be informed of the potential harm to the fetus.

male

Advise males of reproductive potential who have female partners to use effective contraception during treatment with REZUROCK and for at least one week after the last dose of REZUROCK.

Infertility

female

Based on findings from rats, REZUROCK may harm female fertility. Effects on fertility are reversible [see Nonclinical Toxicology (13.1)].

male

Based on findings from rats and dogs, REZUROCK may harm male fertility. Effects on fertility are reversible [see Nonclinical Toxicology (13.1)]. 8.3 Pediatric use

The safety and effectiveness of REZUROCK have been established in pediatric patients 12 years of age and older. The use of REZUROCK in this age group is supported by evidence from adequate and well-controlled studies of REZUROCK in adults, in which additional population pharmacokinetic data indicate that age and body weight do not have a clinically meaningful effect on the pharmacokinetics of the drug substance. Effects, exposure to the drug substance is expected to be similar between adults and pediatric patients over 12 years of age, and the course of illness is sufficiently similar in adult and pediatric patients to allow extrapolation of adult data to pediatric patients.

The safety and effectiveness of REZUROCK in pediatric patients younger than 12 years of age have not been established. 8.4 Use by the elderly

Of the 186 patients with chronic GVHD in the REZUROCK clinical study, 26% were over 65 years old. No clinically meaningful differences in the safety or efficacy of REZUROCK were observed compared with younger patients. 9 instructions

Begrusudil is a kinase inhibitor. The active pharmaceutical ingredient is berusudil mesylate, with a molecular formula of C ₂₇ H ₂₈ N ₆ O ₅ S and a molecular weight of 548.62 g/mol. The chemical name of berusudil mesylate is 2-{3-[4-(1 H -indazol-5-ylamine)-2-quinazolinyl]phenoxy}- N -( Prop-2-yl)acetamide methanesulfonate (1:1). The chemical structure is as follows:

Berusudil mesylate is a yellow powder that is almost insoluble in water, slightly soluble in methanol and DMF, and soluble in DMSO.

REZUROCK lozenges are for oral administration. Each tablet contains 200 mg free base equivalent to

242.5 mg berusudil mesylate. The tablets also contain the following inactive ingredients: microcrystalline cellulose, hypromellose, croscarmellose sodium, colloidal silica, and magnesium stearate.

The tablet film consists of polyvinyl alcohol, polyethylene glycol, talc, titanium dioxide and yellow iron oxide. 10 Clinical Pharmacology 10.1 Mechanism of Action

Begrusudil is an inhibitor of rho-related coiled-coil-containing protein kinase (ROCK). It inhibits ROCK2 and ROCK1 with IC50 values of approximately 100 nM and 3 µM, respectively. Begrusudil downregulates pro-inflammatory responses in ex vivo or in vitro human T cell assays by modulating STAT3/STAT5 phosphorylation and altering Th17/Treg balance. In vitro, berusudil also inhibits aberrant profibrotic signaling. In vivo, berusudil exhibits activity in animal models of chronic GVHD. 10.2 Pharmacodynamics

The exposure-response relationship and time course of the pharmacodynamic response to berusudil have not been determined. 10.3 Pharmacokinetics

Unless otherwise specified, the following pharmacokinetic parameters are presented for patients with chronic GVHD administered 200 mg of berusudil once daily. The mean (coefficient of variation %, CV%) steady-state AUC and C _max of berusudil were 22700 (48%) h·ng/mL and 2390 (44%) ng/mL, respectively. Berusudil C _max and AUC increased in an approximately proportional manner over the dose range of 200 and 400 mg (recommended dose of 1 to 2 times daily). The cumulative ratio for begrusudil is 1.4.

absorb

The median _Tmax of berusudil at steady state was 1.26 to 2.53 hours after administration of 200 mg once daily or twice daily in patients. The mean (CV%) bioavailability of berusudil after a single dose in healthy subjects was 64% (17%).

food effects

In healthy subjects, compared with the fasted state, after administration of a single dose of berusudil with a high-fat and high-calorie meal (800 to 1,000 calories, approximately 50% of total meal calories from fat) , berusudil C _max and AUC increased by 2.2 times and 2 times respectively. Median Tmax was delayed by 0.5 hours.

Distribution

After a single dose of berusuudil in healthy subjects, the geometric mean volume of distribution was 184 L (geo CV% 67.7%).

In vitro, the binding rates of berusudil to human serum albumin and human α1-acid glycoprotein were 99.9% and 98.6%, respectively.

eliminate

In patients, the mean (CV%) elimination half-life of berusudil was 19 hours (39%), and the clearance rate was 9.83 L/hour (46%).

Metabolism

In vitro, berusudil is metabolized primarily by CYP3A4 and, to a lesser extent, CYP2C8, CYP2D6, and UGT1A9.

excretion

After administration of a single oral dose of radiolabeled begrusudil in healthy subjects, 85% of the radioactivity was recovered in the feces (30% unchanged) and less than 5% was recovered in the urine .

special groups

Regarding age (18 to 77 years), sex, weight (38.6 to 143 kg), or mild to moderate renal impairment (eGFR ≥ 60 and < 90 mL/min/ ^1.72m2 to eGFR ≥ 30 and < 60 mL/min /1.72m ² ), no clinically significant differences in berusudil pharmacokinetics were observed. The effect of severe renal impairment on the pharmacokinetics of berusudil has not been studied.

drug interaction studies

Impact of Other Drugs on Berusudil Clinical Studies and Model-Guided Approaches

Strong Cytochrome P450 (CYP) 3A Inhibitor: There was no clinically meaningful effect on berusudil exposure when coadministered with itraconazole in healthy subjects.

Strong CYP3A Inducers: In healthy subjects, coadministration of rifampicin decreased berusudil Cmax by 59% and AUC by 72%.

Moderate CYP3A Inducers: In healthy subjects, coadministration of efavirenz is expected to decrease berusudil Cmax by 32% and AUC by 35%.

Proton Pump Inhibitors: In healthy subjects, coadministration of rabeprazole decreased berusudil Cmax by 87% and AUC by 80%, and omeprazole decreased berusudil Cmax 68% and reduces AUC by 47%.

Effects of berusudil on other drugs

CYP3A Substances: Coadministration of berusudil is expected to increase the _Cmax and AUC of midazolam, a sensitive CYP3A substrate, by approximately 1.3-fold and 1.5-fold, respectively.

CYP2C9 Substrates: Coadministration of berusudil is not expected to have a clinically meaningful effect on the exposure of CYP2C9 substrates such as warfarin.

CYP2C8 Substrates: Coadministration of berusudil is not expected to have a clinically meaningful effect on exposure to CYP2C8 substrates (other than OATP1B1 substrates).

in vitro studies

Transporter system : Berusuudil is a substrate of P-gp. Begrusudil inhibits BCRP, P-gp, and OATP1B1 at clinically relevant concentrations.

Enzyme systems : Berusuudil is an inhibitor of CYP1A2, CYP2C19, CYP2D6, UGT1A1 and UGT1A9. 11Nonclinical Toxicology 11.1 Carcinogenesis, Mutation, Impairment of Fertility

Carcinogenicity studies of berusudil have not been conducted.

Begrusudil was not genotoxic in the in vitro bacterial mutagenicity (Ames) assay, the in vitro chromosomal aberration assay in human peripheral blood lymphocytes (HPBL), or the in vivo rat bone marrow micronucleus assay.

In a combined male and female rat fertility study, berusudil-treated male animals were mated with untreated female animals, or untreated male animals were mated with berusudil-treated Treated female animals were mated. Berusudil was administered orally to male rats at doses of 50, 150, or 275 mg/kg/day 70 days before and throughout the mating period, and 14 days before and until gestation day 7 Orally administered to female rats. Adverse findings in female rats (treated with begrusudil or untreated but mated with treated males) at the 275 mg/kg/day dose included increased pre- or post-implantation loss and viable embryos The quantity decreases. Administration of berusudil to male rats at a dose of 275 mg/kg/day resulted in sperm abnormalities (decreased motility, decreased counts, and increased percentage of abnormal sperm) and testicular/epididymal organ changes (weight loss and degeneration) .

At a dose of 275 mg/kg/day, fertility was reduced in either treated males or females and reached statistical significance in males. Adverse changes in male and female reproductive organs also occurred in general toxicology studies; findings included sperm degeneration in dogs at a berusudil dose of 35 mg/kg/day and in rats at 275 mg/kg/day. Follicle development in the ovaries was reduced at doses of Changes are partially or completely reversed during recovery. The exposure (AUC) in dogs at a dose of 35 mg/kg/day and in rats at a dose of 275 mg/kg/day was 0.5 and 8-9 times the clinical exposure at the recommended dose of 200 mg per day, respectively. 12 Clinical Studies 12.1 Chronic Graft-versus-Host Disease

Study KD025-213 (NCT03640481) is a randomized, open-label, multicenter study of REZUROCK in patients with chronic GVHD who have received 2 to 5 prior lines of systemic therapy and require additional therapy. Patients were excluded from the study if: platelets < 50 × 10 ⁹ /L; absolute neutrophil count < 1.5 × 10 ⁹ /L; AST or ALT > 3 × ULN; total bilirubin > 1.5 × ULN; QTc (F) ＞ 480 ms; eGFR ＜ 30 mL/min/1.73 m ² ; or FEV1 ≤ 39%. 66 patients were treated with REZUROCK 200 mg orally once daily. Concomitant treatment of chronic GVHD with supportive care therapy is allowed. Concomitant treatment with GVHD prophylaxis and standard-of-care system chronic GVHD therapy is allowed as long as the subject has been taking a stable dose for at least 2 weeks prior to the study. Initiation of new systemic chronic GVHD therapies in the study is not permitted.

Demographic and baseline characteristics are summarized in Table 32 .

Table 32: Demographic and Baseline Characteristics of Patients with Chronic GVHD REZUROCK 200 mg once daily (N = 65) Median age (years) (minimum, maximum) 53 (21, 77) Age ≥ 65 years old, n(%) 17 (26) Male, n(%) 42 (65) Race, n(%) white people 54 (83) Black person 6 (9) Other or not reported 5 (8) Median (range) time from chronic GVHD diagnosis (months) 25.3 (1.9, 162.4) Involving ≥ 4 organs, n(%) 31 (48) Median (range) number of prior lines of therapy 3 (2, 6) Number of previous lines of therapy, n(%) 2 23 (35) 3 12 (19) 4 15 (23) ≥ 5 15 (23) Previous treatment of chronic GVHD with ibrutinib, n(%) 21 (32) Previous treatment with ruxolitinib for chronic GVHD, n(%) 20 (31) The last therapy is refractory, n(% ^a ) 43/55 (78) Severe chronic GVHD, n(%) 46 (71) REZUROCK 200 mg once daily (N = 65) Median (range) global severity rating 7 (2, 9) Median (range) Lee Symptom Scale score at baseline 27 (7, 56) Median (range) corticosteroid dose at baseline (PE/kg) ^b 0.19 (0.03, 0.95) ^a Denominator excludes patients with unknown status ^b Prednisone equivalents/kg

REZUROCK's efficacy is based on overall response rate (ORR) through Day 1 of Cycle 7, where ORR includes complete response or partial response according to 2014 NIH response criteria. ORR results are presented in Table 33 . The ORR was 75% (95% CI: 63, 85). The median duration of response, measured from first response to progression, death, or initiation of new systemic therapy for chronic GVHD, was 1.9 months (95% CI: 1.2, 2.9). Median time to first response was 1.8 months (95% CI: 1.0, 1.9). Among patients who achieved a response, 62% (95% CI: 46, 74) were free of death or initiation of new systemic therapy for at least 12 months after response.

Table 33: Overall Response Rate to Cycle 7 Day 1 in Patients with Chronic GVHD in Study KD025-213 REZUROCK 200 mg once daily (N = 65) Overall response rate (ORR) 49 (75%) 95% confidence ^intervala (63%, 85%) full response 4 (6%) partial reaction 45 (69%) ^aEstimated using the Clopper-Pearson method

The ORR results were supported by an exploratory analysis of patient-reported symptom distress, which showed that 52% (95% CI: 40, 65) of patients had a reduction in Lee Symptom Scale total score by Cycle 7 Day 1 At least 7 points. 13How to supply / store and handle

REZUROCK 200 mg tablets are supplied as pale yellow, film-coated, oval-shaped tablets containing 200 mg of berusudil (equivalent to 242.5 mg of berusudil mesylate). Each tablet is debossed with "KDM" on one side and "200" on the other side, and is packaged as follows:

30 200 mg tablets per bottle: NDC 79802-200-30

Store at room temperature 20ºC to 25ºC (68°F to 77°F); short-term travel permitted 15ºC to 30ºC (59°F to 86°F) [see USP Controlled Room Temperature].

Dispense to patients only in original container. Store in original container to protect from moisture. Close the lid tightly after each opening. Do not discard the desiccant. 14Patient consultation information

Patients are advised to read the FDA-approved patient labeling (Patient Information). Embryo-fetal toxicity:

Inform pregnant women and women of reproductive potential of the potential risks to the fetus. Advise females of reproductive potential to inform their healthcare provider of known or suspected pregnancy [ see Warnings and Precautions ( 5.1 ), Use in Specific Populations ( 8.1 , 8.3 ) ].

Advise females of reproductive potential to use effective contraception during treatment with REZUROCK and for at least one week after the last dose [ see Warnings and Precautions ( 5.1 ) ].

Advise males of reproductive potential who have female partners to use effective contraception during treatment with REZUROCK and for at least one week after the last dose [ see Medications in Specific Populations ( 8.3 ) ].

Lactation

Advise women not to breastfeed during treatment with REZUROCK and for at least one week after the last dose [ see Medication in Specific Populations ( 8.2 ) ].

Infertility

Inform males and females of reproductive potential that REZUROCK may impair fertility [ see Medications in Specific Populations ( 8.3 ) ].

invest

Inform patients to take REZUROCK by mouth once daily with food as directed by their doctor and that the oral dose (lozenge) should be swallowed whole with a glass of water at approximately the same time each day and that the lozenge should not be cut, crushed, or chewed [ see Dosage and Administration (2.1)] .

It is recommended that patients who miss a daily dose of REZUROCK should take it as soon as possible on the same day and resume their normal schedule the next day. Patients should not take additional doses to make up for missed doses [ see Dosage and Administration ( 2.1 ) ].

drug interactions

Advise patients to inform their healthcare provider of all concomitant medications, including prescription drugs, over-the-counter drugs, vitamins, and herbal products [ see Drug Interactions ( 7 ) ].

Table 34 : Patient Information Patient Information REZUROCK (REZ-ur-ok) (Berusudil) Lozenges What is REZUROCK ? REZUROCK is a prescription medicine indicated for the treatment of adults and children 12 years of age and older with chronic graft-versus-host disease (chronic GVHD) after they have received at least 2 prior treatments (systemic therapies) and those treatments have failed. It is not known whether REZUROCK is safe and effective in children younger than 12 years old. Before taking REZUROCK , tell your healthcare provider about all your medical conditions, including if you: • Have kidney or liver problems. • Pregnant or planning to become pregnant. REZUROCK may harm your unborn baby. If you are able to become pregnant, your healthcare provider will perform a pregnancy test before starting treatment with REZUROCK. Tell your healthcare provider if you are pregnant or think you may become pregnant during treatment with REZUROCK. o Females of the potential to become pregnant should use effective birth control during treatment with REZUROCK and for at least 1 week after the last dose. o Men whose female partners are capable of becoming pregnant should use effective birth control during treatment with REZUROCK and for at least 1 week after the last dose. • Breastfeeding or planning to breastfeed. It is not known whether REZUROCK passes into breast milk. Do not breastfeed during treatment with REZUROCK and for at least 1 week after the last dose. Tell your health care provider about all the medicines you take , including prescription and over-the-counter medicines, vitamins, and herbal supplements. REZUROCK may affect the way other medicines work, and other medicines may affect how REZUROCK works. Be aware of the medications you take. Keep a list of them and show it to your health care provider and pharmacist when you get new medicines. HOW SHOULD YOU TAKE REZUROCK ? • Take REZUROCK exactly as your healthcare provider tells you. • Do not change your dose or stop taking REZUROCK without first talking with your healthcare provider. • Take REZUROCK once daily with food. • Take REZUROCK at approximately the same time each day. • Swallow REZUROCK tablets whole with a glass of water. • Do not cut, crush or chew REZUROCK tablets. • While you are being treated with REZUROCK, your healthcare provider will do blood tests to check your liver at least once a month. • If you miss a dose of REZUROCK, take it on the same day as soon as you remember. Take your next dose of REZUROCK at a fixed time the next day. Do not take extra doses of REZUROCK to make up for a missed dose. • If you take too much REZUROCK, call your health care provider or go to the nearest hospital emergency room right away. WHAT ARE THE POSSIBLE SIDE EFFECTS OF REZUROCK ? The most common side effects of REZUROCK include: • Infection • Swelling • Tiredness or weakness • Bleeding • Nausea • Stomach (abdominal) pain • Diarrhea • Muscle or bone headache • Shortness of breath • Headache • Cough • High blood pressure If you have certain side effects, you Your healthcare provider may change your REZUROCK dose, temporarily stop, or permanently stop treatment with REZUROCK . REZUROCK may affect male and female fertility. If you are concerned about this, talk to your healthcare provider. These are not all possible side effects of REZUROCK. Call your doctor for medical advice about side effects. You can report side effects to the FDA at 1-800-FDA-1088. You may also report side effects to Kadmon Pharmaceuticals, LLC at 1-877-377-7862. How should REZUROCK be stored ? • Store REZUROCK at room temperature between 68°F and 77°F (20ºC and 25ºC). • Save REZUROCK in its original container. The REZUROCK bottle contains a desiccant packet to help keep the tablets dry (and protected from moisture). Place the desiccant in a bottle. • Close the REZUROCK bottle tightly after taking the dose. Keep REZUROCK and all medicines out of the reach of children. General information about using REZUROCK safely and effectively. Medications are sometimes prescribed for purposes other than those listed in the Patient Information Leaflet. Do not use REZUROCK for unspecified situations. Do not give REZUROCK to others, even if they have the same symptoms as you. This may hurt them. You can ask your pharmacist or health care provider for information about REZUROCK written for health care professionals. What are the ingredients of REZUROCK ? Active Ingredients: Berusudil Mesylate Inactive Ingredients: Lozenge Core: Microcrystalline cellulose, hypromellose, croscarmellose sodium, colloidal silica, and magnesium stearate . Tablet coating: polyvinyl alcohol, polyethylene glycol, talc, titanium dioxide and yellow iron oxide.

While the invention has been described in considerable detail by way of illustration and example for purposes of clarity and understanding, the description and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific documents cited herein are expressly incorporated by reference in their entirety.

without

Claims (31)

2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(prop-2-yl)acetamide, or its pharmaceutical Acceptable salts ( compounds ) for the treatment of chronic graft-versus-host disease (cGVHD) in female patients of reproductive potential include advising said patients to use effective contraception during treatment and for at least one week after the last dose of the compound steps. 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(prop-2-yl)acetamide, or its pharmaceutical Acceptable salts ( compounds ) for the treatment of chronic graft -versus-host disease (cGVHD) in female patients who are pregnant or pregnant at the time the compound is administered, including informing said female patient of the potential risk to the fetus if the compound is administered to the female patient while pregnant . 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide or its pharmaceutically acceptable The salt ( compound ) is accepted for the treatment of chronic graft-versus-host disease (cGVHD) in female patients of reproductive potential, which includes the step of verifying the pregnancy status of said patient before initiating treatment with the compound . The use of claim 1 or 2, further comprising the step of verifying the pregnancy status of the patient before starting treatment with the compound . The use of claim 2, wherein the patient becomes pregnant while receiving treatment with the compound . 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide or its pharmaceutically acceptable salt ( compound ) accepted for the treatment of chronic graft-versus-host disease (cGVHD) in male patients with female partners of reproductive potential, including advising the male patient that during treatment with the compound and at least after the last dose of the compound Steps to use effective birth control within a week. 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide or its pharmaceutically acceptable Salt ( compound ) received for the treatment of chronic graft-versus-host disease (cGVHD) in patients of reproductive potential who use effective contraception during treatment with the compound . The use of claim 7, wherein the patient uses effective contraception during treatment with the compound and for at least one week after the last dose of the compound . The use as described in claim 7, wherein the patient is a female of reproductive potential. The use as described in claim 7, wherein the patient is a male of reproductive potential. 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide or its pharmaceutically acceptable Salt ( Compound ) is accepted for the treatment of chronic graft-versus-host disease (cGVHD) in lactating patients, including advising said patient not to breastfeed during treatment with the compound and for at least one week after the last dose of the compound steps. 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide or its pharmaceutically acceptable The salt ( compound ) received is used to treat chronic graft-versus-host disease (cGVHD) in patients who are not breastfeeding. The use according to any one of claims 1 to 11, wherein the compound is 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy Methanesulfonate salt of ethyl}-N-(propan-2-yl)acetamide. The use of any one of claims 1 to 12, wherein the compound is administered to the patient at a dose of 200 mg per day. The use of any one of claims 1 to 13, wherein the patient suffers from chronic graft-versus-host disease and has experienced failure of at least two previous lines of systemic therapy for the chronic graft-versus-host disease. The use of claim 14, wherein the prior systemic therapy line is selected from the group consisting of prednisone, tacrolimus, ECP, sirolimus, ibrutinib, ruxolitinib, MMF, rituximab , MTX, cyclosporine, imatinib, ixazomib, and ofatumumab. 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide or its pharmaceutically acceptable A salt ( compound ) is accepted for the treatment of chronic graft-versus-host disease (cGVHD) in a patient at risk for pregnancy, which includes the step of informing said patient of the reproductive risks associated with treatment with the compound . The use as described in claim 16, wherein the patient is a female patient with reproductive potential. The use as claimed in claim 17, further comprising the step of verifying the pregnancy status of the patient before starting treatment. The use as described in claim 16, wherein the patient is a male patient. The use of any one of claims 16 to 19, comprising the step of advising said patient to use effective contraception during treatment with the compound and for at least one week after receipt of the last dose of the compound . The use as claimed in any one of claims 16 to 19, comprising the step of informing said patient of the fertility risks associated with treatment with the compound . The use according to any one of claims 16 to 21, wherein the compound is 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy Methanesulfonate salt of ethyl}-N-(propan-2-yl)acetamide. The use of any one of claims 16 to 22, wherein the compound is administered to the patient at a dose of 200 mg per day. The use of any one of claims 16 to 23, wherein the patient suffers from chronic graft-versus-host disease and has experienced failure of at least two previous lines of systemic therapy for the chronic graft-versus-host disease. A kind of 2-{3-[4-(1H-indazol-5-ylamine)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide or its pharmaceutical Use of an acceptable salt ( compound ) in the treatment of graft-versus-host disease in a female patient, comprising the steps of: (a) verifying the pregnancy status of said patient before initiating treatment with the compound ; (b) if said patient inform the patient of the potential risk to the fetus if treated with the compound while pregnant; and (c) advise the patient to use contraception during treatment with the compound and for at least one week after receiving the last dose of the compound . A kind of 2-{3-[4-(1H-indazol-5-ylamine)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide or its pharmaceutical The use of an acceptable salt ( compound ) in the treatment of graft-versus-host disease in a male patient with a female partner of reproductive potential, comprising the steps of: (a) If said patient conceived a partner while receiving treatment with the compound , then Inform the patient of the potential risk to the fetus; and (b) advise the patient to use contraception during treatment with the compound and for at least one week after receiving the last dose of the compound . One uses 2-{3-[4-(1H-indazol-5-ylamine)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide methanesulfonic acid A method of treating chronic graft-versus-host disease (cGVHD) in a patient with SALT (Berusudil), which includes the following steps: (a) Verify that the patient is a reproductive risk patient; and (b) (i) If it is verified that the patient is not a reproductive risk patient, administer berusudil to the patient, or (ii) if the patient is verified to be a reproductive risk patient, inform the patient of the potential reproductive risks of receiving treatment with berusudil. The method of claim 27, wherein in step (a) it is verified that the patient is of reproductive potential, the method further comprising advising the patient that during treatment with berusudil and while receiving its Use effective contraception for at least one week after the last dose. The method of claim 27, wherein in step (a) it is verified that the patient is a lactating patient, the method further comprising advising the patient during treatment with berusudil and after receiving its last dose Do not breastfeed children for at least one week after dose. A method of treating chronic graft-versus-host disease (cGVHD) in a patient, comprising the following steps: (a) Verify that the patient is a reproductive risk patient; and (b) (i) If it is verified that the patient is not a reproductive risk patient, then Administer berusudil to the patient, or (ii) if the patient is verified to be a reproductive risk patient, during treatment with berusudil and for at least one week after the patient receives the last dose Use effective contraception internally.