EA041576B1 - METHODS OF TREATMENT WITH VITAMIN D - Google Patents

Description

Cross-reference to related application

The invention under USC 35, 119(e) claims priority based on US Provisional Application No. 62/314359, filed March 28, 2016, the disclosure of which is incorporated herein by reference.

State of the art

Vitamin D metabolites, known as 25-hydroxyvitamin D ₂ and 25-hydroxyvitamin D ₃ (collectively referred to as 25-hydroxyvitamin D), are fat-soluble steroidal prohormones that help maintain proper levels of the vitamin D hormones, calcium, and phosphorus in the bloodstream. The prohormone 25-hydroxyvitamin D2 is produced from vitamin D2 (ergocalciferol) and 25-hydroxyvitamin _D3 (calcifediol) is produced from vitamin _D3 (cholecalciferol) primarily by one or more liver-localized enzymes. These two prohormones can also be produced outside the liver from vitamin D2 and vitamin _D3 (collectively referred to as vitamin D) in certain cells, such as enterocytes, which contain enzymes identical or similar to those found in the liver.

Vitamin D prohormones are then metabolized in the kidneys by the enzyme CYP27B1 1αhydroxylase to form powerful hormones. The prohormone 25-hydroxyvitamin D2 is metabolized into a hormone known as 1a,25-dihydroxyvitamin D2 (ercalcitriol); in the same way, 25-hydroxyvitamin D ₃ is metabolized to 1a,25-dihydroxyvitamin D ₃ (calcitriol). The production of these hormones from prohormones can also occur outside the kidneys in cells containing the necessary enzyme(s). Rapid increases or excess extracellular concentrations of vitamin D hormones stimulate the expression of the CYP24A1 24-hydroxylase enzyme in cells that contain the vitamin D receptor (VDR). CYP24A1 catabolizes 25-hydroxyvitamin D to form 24,25-dihydroxyvitamin D as the main catabolite.

Vitamin D hormones are essential for human health, performing functions mediated by intracellular vitamin D receptors (VDRs). Vitamin D hormones are involved in the regulation of cell differentiation and growth, parathyroid hormone secretion of parathyroid hormone (PTH), and normal bone formation and metabolism. In particular, vitamin D hormones regulate blood calcium levels by controlling the absorption of dietary calcium and phosphorus by the small intestine and the reabsorption of calcium by the kidneys. Under normal conditions, the effect of vitamin D on the stimulation of calcium absorption in the small intestine is predominant, so that dietary calcium is the main source of serum calcium. However, if dietary calcium or vitamin D is insufficient, then the parathyroid gland increases PTH production to increase calcium mobilization from the bones to maintain serum calcium levels. Excess levels of the hormone vitamin D, whether temporary or prolonged, can lead to side effects, including abnormally high levels of calcium in the urine (hypercalciuria), calcium in the blood (hypercalcemia), phosphorus in the blood (hyperphosphatemia), and adynamic bone disease. Vitamin D hormones are also essential for the normal functioning of the musculoskeletal, immune, and renin-angiotensin systems. Many other functions of vitamin D hormones have been postulated and investigated based on the documented presence of intracellular VDRs in almost all human tissues.

Left untreated, vitamin D deficiency can cause severe bone disorders, including rickets and osteomalacia, and may contribute to the development of many other disorders, including osteoporosis, non-traumatic fractures in the spine and hips, obesity, diabetes, muscle weakness, immunodeficiency, hypertension, psoriasis and various types of cancer.

The Institute of Medicine (IOM) of the National Academy of Sciences concluded that adequate absorption (AI) of vitamin D for a healthy individual ranges from 200 to 600 IU per day, depending on the age and sex of the individual (Standing Committee on the Scientific Evaluation of Dietary Reference Intakes, Dietary reference intakes: calcium, phosphorus, magnesium, vitamin D, and fluoride Washington, DC: National Academy Press (1997) incorporated by reference). Vitamin D AI was determined primarily based on the serum concentration of 25-hydroxyvitamin D sufficient to prevent rickets or osteomalacia caused by vitamin D deficiency (or greater than or exactly 11 ng/mL). The IOM also established a maximum tolerated intake (UL) level for vitamin D, 2000 IU per day, based on evidence that higher doses are associated with an increased risk of hypercalciuria, hypercalcemia, and related effects, including cardiac arrhythmias, seizures, and generalized vascular calcification and other soft tissues. The tolerable upper limit for serum 25-hydroxyvitamin D in healthy subjects is approximately 100 ng/mL or 250 nmol/L (see e.g. Jones, G, Am. J. Clin. Nutr. 88(suppl): 582S-6S, 2008; Holick, MF, Ann Epidemiol 19(2): 73-78, 2009).

However, the definition of vitamin D deficiency (VDI) in general, and in particular in subjects suffering from chronic kidney disease (CKD), is ambiguous. The IOM defines VDI as total 25-hydroxyvitamin D less than 20 ng/mL, while the Kidney Disease Outcomes Quality Initiative (KDOQI), Kidney Disease Improving Global Outcomes, KDIGO) and clinical practice guidelines in endocrinology define VDI as 25-hydroxyvitamin D less than 30 ng/mL (National Kidney Foundation KDOQI Guidelines, 2003; National Kidney Foundation KDIGO Guidelines, 2009; Holick et al., J Clin Endocrinol Metab 96(7):1911-30, 2011). The appearance of the 25hydroxyvitamin D catabolite (25(OH)D3), 24,25-dihydroxyvitamin D (24,25(OH) ₂ D), may be an appropriate measure of sufficiency if the VDI is determined by the ratio of 25(OH)D ₃ :24, 25 (OH) ₂ D ₃ is greater than 20 (Kaufmann et al., J Clin Endocrinol Metab 99(7):2567-2574, 2014). Serum levels of 25-hydroxyvitamin D and 24,25dihydroxyvitamin D are closely correlated, with increased catabolism of 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D with increased levels of 25-hydroxyvitamin D in normal (i.e., non-CKD) groups (Berg et al., Clin Chem 61(6):877-884, 2015; Wagner at al., J Ster Biochem Mol Bio 126:7222, 2011). Although CYP24 is usually reduced in normal subjects with VDI, elevated levels of CYP24 are observed in subjects with CKD (Helvig et al., Kidney Int 78(5):463-72, 2010; International Patent Application No. PCT/US2009/39355 ).

Regardless of how VDI is determined, traditional oral vitamin D supplements are far from ideal for achieving and maintaining optimal blood levels of 25-hydroxyvitamin D. These formulations typically contain 400 to 5000 IU of vitamin D3 or 50,000 IU of vitamin D ₂ and are formulated for rapid or immediate release in the gastrointestinal tract. When given consistently at high doses, which is often necessary for vitamin D saturation in CKD, these products have significant, and often severe, limitations. In addition, prior use of immediate release formulations of calcifediol has not been shown to be effective in suppressing elevated PTH levels in clinical studies conducted in patients with stage 3 or 4 CKD.

Controlled-release oral formulations and slow-acting (eg, slow intravenous bolus) formulations of 25-hydroxyvitamin D ₂ and/or 25-hydroxyvitamin D3 for parenteral administration can be administered to treat 25-hydroxyvitamin D insufficiency and deficiency without superphysiological fluctuations in intraluminal , intracellular and blood levels of 25-hydroxyvitamin D and their effects; without a significant increase in the catabolism of the introduced 25-hydroxyvitamin D; and without the severe side effects associated with vitamin D supplementation. Timed release controlled release formulations are effective in lowering PTH levels without undesirable increases in serum calcium and phosphorus levels and are thus suitable for the treatment of secondary hyperparathyroidism, for example, in patients with CKD ; see international patent applications no.

Controlled release formulations provide substantially improved absorption of 25-hydroxyvitamin D via transport on the vitamin D binding protein (DBP) and reduced absorption via transport in chylomicrons. These compositions also provide substantially constant blood concentrations of 25-hydroxyvitamin D over a 24 hour post-dose period. By providing gradual, sustained and direct release of 25-hydroxyvitamin D2/25-hydroxyvitamin D3 and absorption predominantly into circulating DBP (rather than chylomicrons), blood, intraluminal and intracellular concentrations of 25hydroxyvitamin D increase dramatically, i.e. it is possible to eliminate or exclude supra-physiological levels and the corresponding undesirable catabolism. In addition, by providing gradual and sustained release, serum concentrations of 25-hydroxyvitamin D can be increased and maintained in a more predictable manner than with immediate release formulations, allowing an appropriate dose to be administered and reducing or eliminating the need for frequent patient monitoring.

The essence of the invention

The present disclosure relates to methods of treating vitamin D-responsive diseases by increasing serum levels of 25-hydroxyvitamin D (e.g., total 25-hydroxyvitamin D or calcifediol) to high levels while simultaneously increasing levels of 1,25-dihydroxyvitamin D (e.g., total 1, serum 25-dihydroxyvitamin D or calcitriol) to high levels without hypercalcemia.

The present disclosure also relates to methods for treating vitamin D deficiency and secondary hyperparathyroidism in patients with chronic kidney disease (CKD). In one aspect, a method of treating secondary hyperparathyroidism in a CKD patient comprises administering to said patient repeated doses of 25-hydroxyvitamin D effective to increase the patient's serum 25-hydroxyvitamin D level to greater than 90 ng/mL. In another aspect, a method of treating vitamin D deficiency in a patient with CKD comprises administering to said patient repeated doses of 25-hydroxyvitamin D effective to control the ratio of 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D in the patient's serum to less than 20. In another aspect, the method treatment of secondary hyperparathyroidism in a patient with CKD comprises administering to said patient repeated doses of 25-hydroxyvitamin D effective to increase the patient's serum 25-hydroxyvitamin D level to a value greater than 90

- 2 041576 ng / ml to control the ratio of 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D in the patient's serum to a value of less than 20.

The present disclosure also relates to methods of administering a sustained release formulation of 25hydroxyvitamin D, optionally containing a controlled release waxy agent, an emulsifier, an absorption enhancer, an oleaginous carrier, and a stabilizing agent; to a patient in need thereof, comprising administering to the subject repeated doses of an extended release oral dosage form effective to achieve one or _more of the following: /ml; (2) Tmax 25-hydroxyvitamin D ₃ adjusted for baseline from about 5 days to about 60 days; (3) AUC0-6 _{weeks of} 25-hydroxyvitamin D ₃ adjusted for baseline from about 100 to about 3300 ng-d/ml; (4) Cmax 1,25 dihydroxyvitamin D adjusted for baseline from about 0.1 pg/ml to about 50 mg/ml; (5) Tmax 1,25-dihydroxyvitamin D adjusted for baseline from about 1 day to about 44 days; (6) AUC0.6 _{weeks of} 1,25-dihydroxyvitamin D adjusted for baseline from about 1 to about 1300 g-d/ml; (7) increase in mean serum 25-hydroxyvitamin D from about 1 to 7 ng/mL/week; (8) an increase in mean serum 1,25-dihydroxyvitamin D from about 1 to about 10 pg/mL/week; (9) an average constant serum level of 25-hydroxyvitamin D from about 50 to about 55 ng/ml; (10) decrease in intact plasma PTH from about 30 to about 80 pg/ml from baseline; (12) a constant serum 25-hydroxyvitamin D level of greater than about 50 to about 100 ng/mL; (13) an increase in plasma 1,25-dihydroxyvitamin D of at least about 10 pg/mL from baseline; (14) an increase in serum calcium from 0 to about 0.3 mg/dL from baseline; (15) an increase in serum phosphorus from 0 to about 0.3 mg/dL from baseline; (16) a decrease in serum BSAP of at least about 10 U/L from baseline; (17) a decrease in serum CTX-1 of at least about 100 pg/ml from baseline; (18) a decrease in serum P1NP of at least about 30 ng/mL from baseline; and (19) decrease in plasma iPTH from about 15% to about 40% of baseline.

In any of the methods described herein, the patient does not necessarily have stage 1, 2, 3, 4, or 5 CKD. In various embodiments, said method comprises administering repeated doses of 25-hydroxyvitamin D in an amount effective to increase the patient's serum 25-hydroxyvitamin D level to greater than 100, greater than 125, greater than 150, greater than 175, or greater than 200 ng/mL without causing hypercalcemia. , hyperphosphatemia and/or hypercalciuria. Said method may include administering repeated doses to achieve a serum 25-D level of about 120 to about 200 ng/mL, or about 120 to about 160 ng/mL, or about 150 to about 200 ng/mL, for example, to patients with stage 5 CKD on dialysis. Optionally, the method of the present disclosure comprises administering repeated doses of 25-hydroxyvitamin D in an amount effective to control the ratio of 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D in the patient's serum to less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, or less than 10.

For the methods described herein, it is contemplated that optional features, including but not limited to components, their ranges, substituents, conditions, and steps, may be selected from various aspects, embodiments, and examples provided herein. Additional aspects and advantages will become apparent to those skilled in the art upon reading the following detailed description taken in conjunction with the drawings. Although various forms of embodiments can be applied to these methods, the following description includes specific implementations with the understanding that this description is illustrative and is not intended to limit the present invention to the specific implementations described herein.

Brief description of graphic materials

In FIG. 1A shows the mean (±CO) change in iPTH from baseline in plasma of subjects treated with placebo (open circles) or extended release 25-hydroxyvitamin D ₃ formulation (solid circles) for 26 weeks. In FIG. 1B shows the mean (±CO) change in iPTH from baseline in plasma of stage 3 CKD subjects treated with placebo (open circles), stage 3 CKD subjects treated with extended release 25-hydroxyvitamin D ₃ formulation (open circles). squares), stage 4 CKD subjects treated with placebo (solid circles), and stage 4 CKD subjects treated with extended release 25-hydroxyvitamin D ₃ formulation (solid squares) for 62 weeks. In FIG. 1C shows mean (±CO) plasma iPTH levels (pg/mL) compared with total 25-hydroxyvitamin D serum levels (ng/mL) in subjects with stage 3 or 4 CKD (top), with stage 3 CKD (middle). diagram) or with CKD stage 4 (lower diagram). Asterisks indicate statistical significance relative to the group with serum total 25-hydroxyvitamin D 0-20 ng/mL.

- 3 041576

In FIG. 2A shows mean (±CO) calcifediol (ng/mL) serum levels of subjects treated with placebo (squares) or sustained release 25-hydroxyvitamin D ₃ formulation (diamonds) for 26 weeks. Triple asterisks (***) denote significant difference relative to placebo, p < 0.0001. In FIG. 2B shows mean (±CO) serum levels of total 1,25-dihydroxyvitamin D (pg/mL) compared to total 25-hydroxyvitamin D serum levels (ng/mL) in subjects with stage 3 or 4 CKD (upper chart), with CKD 3 stages (middle chart) or with CKD 4 stages (bottom chart). Asterisks indicate statistical significance relative to the group with serum total 25-hydroxyvitamin D 0-20 ng/mL.

In FIG. 3A shows the mean (±CO) serum levels of 24,25-dihydroxyvitamin D ₃ (ng/mL) of subjects treated with placebo (squares) or extended release 25-hydroxyvitamin D ₃ formulation (diamonds) for 26 weeks. Triple asterisks (***) denote significant difference relative to placebo, p < 0.0001. In FIG. 3B shows the ratio of serum calcifediol (25-hydroxyvitamin D ₃ ) to sera 24,25-dihydroxyvitamin D ₃ in subjects treated with placebo (squares) or sustained release formulation of 25-hydroxyvitamin D3 (diamonds) for 26 weeks. Asterisks indicate significant difference relative to placebo (*=p < 0.05, **=p < 0.001, ***=p < 0.0001). In FIG. 3C and 3D show the ratio of calcifediol to 24,25-dihydroxyvitamin D3 versus serum calcifediol at baseline (Figure 3C) and at the end of the EAP (Figure 3D) for patients with stage 3 and 4 CKD, who were given placebo for 26 weeks. In FIG. 3E and 3F show the ratio of calcifediol to 24,25-dihydroxyvitamin D ₃ versus serum calcifediol at baseline (Fig. 3E) and at the end of the efficacy assessment (EAP) period (Fig. 3F) for patients with stage 3 and 4 CKD. who were administered a sustained release formulation of 25-hydroxyvitamin D ₃ for 26 weeks.

In FIG. 4A-4C shows the ratio of calcifediol to 24,25-dihydroxyvitamin D ₃ compared to serum calcifediol levels at visit 6/day 57 (FIG. 4A), visit 10/day 141 (FIG. 4B), and visit 13 /at day 183 (FIG. 4C) for patients with stage 3 CKD who were administered a sustained release formulation of 25-hydroxyvitamin D3 for 26 weeks.

In FIG. 5A-5C show the ratio of calcifediol to 24,25-dihydroxyvitamin D3 compared to serum calcifediol levels at visit 6/day 57 (FIG. 5A), visit 10/day 141 (FIG. 5B), and visit 13/day. at day 183 (FIG. 5C) for patients with stage 4 CKD who were administered a 25-hydroxyvitamin D ₃ extended release formulation for 26 weeks.

In FIG. 6A shows the mean (±CO) serum calcium (required value < 9.8 mg/dl) and serum phosphorus (required value < 5.5 mg/dl) of subjects administered placebo (squares) or formulation 25- hydroxyvitamin D ₃ extended release (diamonds) for 26 weeks. In FIG. 6B shows mean (±CO) serum calcium (mg/dL) and FIG. 6C shows mean (±CO) serum phosphorus compared to serum total 25-hydroxyvitamin D. In FIG. 6D shows the mean (±CO) urinary calcium/creatinine ratio (Ca/Cr) and FIG. 6E shows urinary mean tubular reabsorption of phosphate (TRP) compared with serum levels of total 25-hydroxyvitamin D in patients with stage 3 and 4 CKD treated with a sustained release formulation of 25-hydroxyvitamin D ₃ for 26 weeks.

In FIG. 7A shows mean (±CO) serum levels of bone-specific alkaline phosphatase (BSAP) (U/L) versus total 25-hydroxyvitamin D serum levels (ng/mL) of subjects with stage 3 or 4 CKD (upper panel) , with stage 3 CKD (middle chart) or with stage 4 CKD (bottom chart). In FIG. 7B shows the mean (±CO) serum levels of cross-linked Collagen Type 1 Ctelopeptide (CTX-1) (pg/mL) (top chart) and FIG. 7C shows mean (±CO) serum levels of N-terminal procollagen type 1 (P1NP) (ng/mL) (lower panel) compared to total 25-hydroxyvitamin D serum levels (ng/mL) in subjects with CKD 3 or 4. stages. Asterisks indicate statistical significance relative to the group with serum total 25-hydroxyvitamin D 0-20 ng/mL.

In FIG. 8 and 9 are serum calcifediol, serum calcitriol, serum calcium, serum phosphorus, and 24,25-dihydroxyvitamin D3 serum levels obtained by administering increasing doses of sustained-release oral calcifediol to subjects over a period of up to one year. .

Detailed description

The present disclosure relates to methods of administering vitamin D compounds and to methods of treating diseases susceptible to vitamin D.

The present disclosure also relates to methods for treating vitamin D deficiency and secondary hyperparathyroidism in patients with chronic kidney disease (CKD). In various embodiments, these methods are used to treat patients with stage 1, stage 2, stage 3, stage 4, or stage 5 CKD, or a mixed group of patients with a combination of any of the above stages. In one

- 4 041576 aspect, the method according to the present description is used to treat a patient with stage 3 or 4 CKD.

In another aspect, the method of the present disclosure is used to treat a patient with stage 5 CKD, such as a patient on hemodialysis. Optionally, the CKD patient has a serum total 25hydroxyvitamin D concentration of less than 30 ng/mL.

The following definitions may be useful to facilitate understanding of the present description by experienced practitioners. Unless otherwise defined in this context, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art.

In this context, the term containing means the possible inclusion of other agents, elements, steps or features, in addition to those specifically indicated.

In this context, the term 25-hydroxyvitamin D refers to one or more of 25hydroxyvitamin _D2 , 25hydroxyvitamin _D3 (also known as calcifediol, calcidiol and 25hydroxycholecalciferol), 25hydroxyvitamin _D4 , 25hydroxyvitamin _D5 , 25hydroxyvitamin D7 , analogues of the above compounds and their combinations. In particular, it is contemplated that in any embodiment described herein, 25-hydroxyvitamin D may include 25hydroxyvitamin D ₃ , 25-hydroxyvitamin D2, or a combination of 25-hydroxyvitamin D ₃ and 25hydroxyvitamin D ₂ . For example, it is specifically contemplated that in any embodiment described herein, the 25-hydroxyvitamin D may include 25-hydroxyvitamin D ₃ . Serum 25-hydroxyvitamin D and total 25-hydroxyvitamin D serum refers to total 25-hydroxyvitamin D ₂ and 25-hydroxyvitamin D ₃ as measured in serum by laboratory analysis, unless specific reference is made to a specific form of 25hydroxyvitamin D, e.g. 25-hydroxyvitamin D3.

As used herein, the term 1,25-dihydroxyvitamin D refers to one or more of 1,25-dihydroxyvitamin D2, 1,25-dihydroxyvitamin D3, 1,25-dihydroxyvitamin D4, 1,25-dihydroxyvitamin _D5 , 1,25-dihydroxyvitamin D7, analogs of the foregoing. compounds and their combinations. For example, 1,25-dihydroxyvitamin D may include 1,25-dihydroxyvitamin D ₂ , 1,25-dihydroxyvitamin D ₃ , or a combination of 1,25-dihydroxyvitamin D ₂ and 1,25-dihydroxyvitamin D ₃ . Serum 1,25-dihydroxyvitamin D and total serum 1,25-dihydroxyvitamin D are to be understood as total 1,25-dihydroxyvitamin _D2 and 1,25lihydroxyvitamin _D3 as measured in serum by laboratory analysis unless specific reference is made. to a specific form of 1,25-dihydroxyvitamin D.

As used herein, the term vitamin D supplementation therapy refers to administering to a patient one or more vitamin D prehormones (eg, cholecalciferol or ergocalciferol) or analogues thereof, or a vitamin D prohormone (eg, 25-hydroxyvitamin D2 or 25-hydroxyvitamin D3) or analogues thereof. Examples of compounds suitable for use in vitamin D supplementation therapy include ergocalciferol, cholecalciferol, 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3.

As used herein, the term vitamin D hormone replacement therapy refers to administering to a patient an effective amount of one or more of active vitamin D hormones, which include active vitamin D hormone metabolites, and active vitamin D hormone analogs, including 1α-hydroxylated vitamin D compounds. Suitable for use in vitamin D hormone replacement therapy include the vitamin D hormones 1,25dihydroxyvitamin D ₂ , 1,25-dihydroxyvitamin D ₃ , 1,25-dihydroxyvitamin D ₄ and their analogs, including doxercalciferol and paricalcitol.

As used herein, co-administration refers to the administration of two or more compounds, for example more than one vitamin D supplementation compound, or a vitamin D supplementation compound and a vitamin D hormone replacement compound, to the same patient. For example, co-administration includes (a) simultaneous administration of the first and second compound, and (b) administration of the first compound followed by administration of the second compound. For example, the first and second compounds can be administered within 24, 8, 4, 2, or 1 hours of each other. In other embodiments, you can use other time intervals between the introduction of the first and second compounds.

As used herein, the terms controlled release, modified release, sustained release, and sustained release refer to the release of the administered 25-hydroxyvitamin D compound from the composition in a manner other than immediate release. For example, a sustained release oral dosage form ensures that 25-hydroxyvitamin D is available for an extended period of time after ingestion due to sustained release dosage form design (see USP 24 <1151>).

The above terms do not necessarily include delayed release characteristics where the release of 25-hydroxyvitamin D from the oral dosage form occurs only after the passage of the capsule through the stomach (see USP 24 <1151>).

In this context, the term hypercalcemia refers to the pathological condition of the patient,

- 5 041576 in which two consecutive measurements of the patient's serum calcium during the course of treatment (for example, within one month or two weeks apart) have values of more than about 10.3 mg/dl.

As used herein, the term hyperphosphatemia refers to the pathological condition of a patient in which two consecutive measurements of serum phosphorus during a course of treatment (eg, within one month or two weeks apart) have values greater than about 5.5 mg/dL.

In this context, the term hypercalciuria refers to a pathological condition in which the ratio of urinary calcium to urinary creatinine (Ca/Cr) of the patient is > 0.2.

In this context, the term bioequivalent refers to a dosage form that is biologically equivalent in vivo to the dosage form described herein if the 90% confidence intervals (90% CI) of the Cmax and AUC(o- _in f) ratios between the bioequivalent dosage form and the reference dosage form form are in the range from 80 to 125% (0.8-1.25) when tested in equivalent ways (including equivalent doses and conditions on an empty stomach).

In particular, it should be understood that any numerical value specified herein includes all values from the lower value to the upper value, i.e. all possible combinations of numerical values between the smallest specified value and the largest specified value should be understood as being explicitly stated in this application. For example, if a concentration range or a preferred effect range is specified as being 1 to 50%, then it is contemplated that values such as 2 to 40%, 10 to 30%, or 1 to 3% are explicitly listed herein, and etc. In another example, said concentration of about 20% includes values from 19.5% to 20.5%. These are just a few examples of what is specifically provided.

In a jurisdiction that prohibits patenting methods practiced on the human body, the meaning of administering a composition to a human is limited to administering a controlled substance that the human will self-administer in some way (e.g., orally, by inhalation, topical application, injection, insertion, etc.) , as well as the manufacture of a medicinal product for use in the methods described in this document. The broadest reasonable interpretation is provided that is consistent with the laws or regulations that define patentable subject matter. In a jurisdiction that does not prohibit patenting methods practiced on the human body, administration of the compositions includes methods practiced on the human body as well as the foregoing.

Compositions and methods according to the present invention are suitable for the prophylactic or therapeutic treatment of diseases susceptible to vitamin D, i. diseases in which vitamin D, 25-hydroxyvitamin D or active vitamin D (for example, 1,25-dihydroxyvitamin D) prevents the onset or progression of the disease, or reduces the signs or symptoms of the disease. Such diseases susceptible to vitamin D include cancer (eg, breast, lung, skin, melanoma, colon, colon, rectal, rectal, prostate, and bone cancers). 1,25-dihydroxyvitamin D has been found to induce cell differentiation and/or inhibit cell proliferation in vitro for many cells. Diseases susceptible to vitamin D also include autoimmune diseases such as type I diabetes, multiple sclerosis, rheumatoid arthritis, polymyositis, dermatomyositis, scleroderma, fibrosis, Graves' disease, Hashimoto's disease, acute or chronic graft rejection, acute or chronic graft versus host disease. , inflammatory bowel disease, Crohn's disease, systemic lupus erythematosus, Sjögren's syndrome, eczema and psoriasis, dermatitis including atopic dermatitis, contact dermatitis, allergic dermatitis and/or chronic dermatitis. Diseases susceptible to vitamin D also include other inflammatory diseases such as asthma, chronic obstructive pulmonary disease, polycystic kidney disease, polycystic ovary syndrome, pancreatitis, nephritis, hepatitis, and/or infections. Diseases susceptible to vitamin D have also been described to include hypertension and cardiovascular disease. Thus, the present invention provides for the prophylactic or therapeutic treatment of subjects at risk of or suffering from cardiovascular disease, e.g., subjects with atherosclerosis, arteriosclerosis, coronary artery disease, cerebrovascular disease, peripheral vascular disease, myocardial infarction, myocardial ischemia, cerebral ischemia. , stroke, congestive heart failure, cardiomyopathy, obesity or other weight disorders, lipid disorders (eg, hyperlipidemia, dyslipidemia, including associated diabetic dyslipidemia and mixed dyslipidemia, hypo-alpha-lipoproteinemia, hypertriglyceridemia, hypercholesterolemia, and low HDL (high-density lipoprotein) levels) ), metabolic disorders (eg, metabolic syndrome, type II diabetes mellitus, type I diabetes mellitus, hyperinsulinemia, impaired glucose tolerance, insulin resistance, diabetic complications, including neuropathy, nephropathy, retinopathy, diabetic foot ulcer and cataracts) and/or thrombosis.

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Diseases in which modulation of vitamin D compound levels may be beneficial include, but are not limited to: (i) in the parathyroid gland, hypoparathyroidism, pseudohypoparathyroidism, secondary hyperparathyroidism; (ii) in the pancreas, diabetes; (iii) in the thyroid gland, medullary carcinoma; (iv) in the skin psoriasis; wound healing; (v) in the lungs, sarcoidosis and tuberculosis; (vi) in the kidneys - chronic kidney disease, hypophosphatemic vitamin D-resistant rickets (VDRR), vitamin D-dependent rickets; (vii) in bones - anticonvulsant treatment, imperfect bone fibrogenesis, fibrous generalized osteodystrophy, osteomalacia, osteoporosis, osteopenia, osteosclerosis, renal osteodystrophy, rickets; (viii) in the intestine - glucocorticoid antagonism, idiopathic hypercalcemia, malabsorption syndrome, steatorrhea, tropical sprue; and (ix) autoimmune disorders.

In various embodiments of the present invention, the diseases in which modulating levels of vitamin D compounds may be beneficial are selected from cancer, dermatological disorders (e.g., psoriasis), parathyroid disorders (e.g., hyperparathyroidism and secondary hyperparathyroidism), bone disorders (e.g., osteoporosis) and autoimmune disorders.

In one type of embodiments, the patient suffers from osteoporosis. The patient may be suffering from postmenopausal osteoporosis. The patient may be suffering from senile osteoporosis.

In one type of embodiment, the patient is a patient already treated with a bisphosphonate. For example, the bisphosphonate may be etidronate, pamidronate, alendronate, risedronate, zolendronate, or ibandronate. The bisphosphonate may be a bisphosphonate having a long bone half-life, such as at least 3 years or at least 5 years.

In one aspect, the present disclosure provides a method of treating a vitamin D responsive disease by elevating a patient's serum 25-hydroxyvitamin D level as described herein while simultaneously elevating a patient's serum 1,25-dihydroxyvitamin D level to the high level described herein, without hypercalcemia. For example, the serum level of 25-hydroxyvitamin D in a patient can be increased using an extended release oral dosage form containing 25hydroxyvitamin D ₃ , 25hydroxyvitamin D _{2 ,} or a combination of 25hydroxyvitamin D ₃ and 25hydroxyvitamin D2.

Repeated doses of 25-hydroxyvitamin D can be administered, for example, daily, every other day, two or three times a week, every week, every other week, or once a month. Optionally, repeated doses of 25-hydroxyvitamin D are administered over an extended period, such as for at least one week, at least two weeks, at least three weeks, one month, at least two months, at least three months. at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, at least one year or more. It is contemplated that an escalating dose schedule may be used in which repeated doses are administered on the first schedule for a first period of time, and then at a higher dose on the same schedule for a second period of time. For example, the dose may be an initial dose given daily for one month, then an increased dose given daily for a second month, then an even higher dose given daily for a third month. In another embodiment, the dose may be an initial dose given 3 times a week for one month, followed by an increased dose given 3 times a week for a second month, and then an even higher dose given 3 times a week. during three months.

In one type of embodiments, 25-hydroxyvitamin D (eg, calcifediol) can be administered on an empty stomach. In one type of embodiment, 25-hydroxyvitamin D (eg, calcifediol) may be administered at bedtime.

In one aspect, 25-hydroxyvitamin D can be administered by controlling the patient's daily intake of dietary calcium (combined from food sources and supplements) at 1000, or less than 1000, or less than 900, or less than 800, or less than 700, or less than 600, or less than 500 mg/day, such as 400-1000 or 400-900 or 400-800 or 400-700 or 400-600 or 500-700 mg/day.

The methods of the present disclosure are effective in increasing a patient's serum 25-hydroxyvitamin D levels to levels previously considered unsafe, but without causing one or more side effects associated with vitamin D toxicity. Recent data suggest that the target 25-hydroxyvitamin D level in serum in the treatment of patients with CKD is from 40 to 50 ng/ml to lower PTH levels (Ennis et al., J Nephrol 29(1):63-70, 2015). While serum levels of 25-hydroxyvitamin D at 100 ng/mL are considered to be acceptable upper limits of normal, the methods of the present disclosure can be used to increase serum levels of 25-hydroxyvitamin D to significantly higher levels without causing, for example, one or more of hypercalcemia, hyperphosphatemia, hypercalcinuria, and adynamic bone disease. In one aspect, said method is effective to increase a patient's serum 25-hydroxyvitamin D concentration to greater than about 100 ng/ml, such as greater than about 110, greater than about 120, greater than about 130, greater than about 140, greater than about 150, greater than about 160, greater than about 170, greater than about 180, greater than about 190, greater than about 200, greater than about 210, greater than about 220, greater than about 230, greater than about 240, greater than about 250, greater than about 300, or greater than about 250 ng/mL. In another aspect, said method is effective for increasing the level of 25-hydroxyvitamin D in the patient's serum to 500, to 450, to 400, to 350, to 300, to 250 ng/ml, or, for example, to 240, or to 230, or to 220 , or up to 210, or up to 200, or up to 190, or up to 180 ng/ml. Optionally, the concentration of 25-hydroxyvitamin D in the patient's serum is increased to about 90 to about 120 ng/ml, or about 100 to about 150 ng/ml, or about 120 to about 160 ng/ml, or about 140 to about 180 ng/ml, or about 150 to about 200 ng/ml, or about 180 to about 200 ng/ml, or about 190 to about 220 ng/ml, or about 150 to about 250 ng/ml, or from about 200 to about 300 ng/ml, or from about 250 to about 300 ng/ml, or from about 150 to about 300 ng/ml.

Vitamin D prohormones 25-hydroxyvitamin D ₃ and 25-hydroxyvitamin D ₂ are metabolized primarily in the kidneys by CYP27B1 1a-hydroxylase to potent, active hormones (25-hydroxyvitamin D2 is metabolized to 1a,25-dihydroxyvitamin D2 and 25-hydroxyvitamin D ₃ is metabolized to 1a ,25-dihydroxyitamine D ₃ ). When administered in repeated doses of extended-release oral 25hydroxyvitamin D ₃ to patients with stage 3 and 4 CKD, a dose of 30 µg/day has been found to provide an increase in serum 1,25-dihydroxyvitamin D, but a dose of 60 µg/day provides 1. 25-dihydroxyvitamin D in serum, which is approximately equal to its content at a dose of 30 mcg / day; in addition, a dose of 90 mcg/day does not lead to a further increase in the content of 1,25-dihydroxyvitamin D in serum. At lower doses, 1,25-dihydroxyvitamin D is believed to be produced primarily by renal 1-hydroxylase, but as serum levels of 1,25-dihydroxyvitamin D increase, renal 1-hydroxylase is suppressed. It is believed that by increasing the dose of sustained release oral 25-hydroxyvitamin D3 (eg, 90 μg/day), extrarenal production of 1,25-dihydroxyvitamin D is more likely to increase serum levels of 1,25-dihydroxyvitamin D.

It has also been observed that by administering increasing repeated doses of sustained release oral 25-hydroxyvitamin D ₃ , serum levels of 1,25-dihydroxyvitamin D can be raised to very high levels without causing hypercalcemia. It is believed that a gradual increase in serum 25-hydroxyvitamin D and a gradual increase in serum 1,25-dihydroxyvitamin D provide the possibility of physiological adaptation in which absorption of calcium in the intestine does not occur or occurs to a much lesser extent, compared with methods in which the content of 25- serum hydroxyvitamin D and/or serum 1,25 dihydroxyvitamin D increase more rapidly.

In another aspect, the present disclosure provides a method of treating secondary hyperparathyroidism in a patient with CKD, comprising administering repeated doses of 25-hydroxyvitamin D to said patient effective to increase the patient's serum 25-hydroxyvitamin D level to greater than 90 ng/mL. In another aspect, the present disclosure provides a method for treating secondary hyperparathyroidism in a patient with CKD, comprising administering to said patient repeated doses of 25-hydroxyvitamin D effective to increase the patient's serum 25-hydroxyvitamin D level to greater than 90 ng/mL and to regulate the 25- hydroxyvitamin D to 24,25-dihydroxyvitamin D in the patient's serum to a value of less than 20 or less than 15.

In another aspect, the method of the present disclosure comprises administering repeated doses of a vitamin D supplementation compound, e.g., 25-hydroxyvitamin D and/or ergocalciferol or cholecalciferol, in an amount effective to control the ratio of 25hydroxyvitamin D to 24,25-dihydroxyvitamin D in the patient's serum to less than 20, or less than 18, or less than 16, or less than 15, or less than 14, or less than 12, or less than 10. repeated doses of 25-hydroxyvitamin D effective to control the ratio of 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D in the patient's serum to less than 20. In another aspect, the present disclosure provides a method of treating vitamin D deficiency in a patient with CKD, comprising administering said repeated doses of a compound for vitamin D supplementation, e.g., 25-hydroxy vitamin D and/or ergocalciferol or cholecalciferol, in an amount effective to increase the level of 25hydroxyvitamin D in the patient's serum to a value of more than about 100 ng/ml, and to control the ratio of 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D in the patient's serum to values less than 20. In healthy subjects, a plot of the ratio of 25-hydroxyvitamin D to

- 8 041576

24,25-dihydroxyvitamin D (25(OH)D3:24.25(OH)2D3) in serum from 25hydroxyvitamin D (25(OH)D3) in serum reaches a plateau if 25(OH)D3:24.25( OH)2D3 is less than 20, indicating vitamin D sufficiency (Kaufmann et al., supra). In CKD patients, a 25(OH)D3:24.25(OH)2D3 ratio of less than 25 was not achieved even though serum 25hydroxyvitamin D levels approached and exceeded 100 ng/mL (Fig. 3F), indicating that the ratio of 25(OH)D3:24.25(OH)2D3 corresponding to vitamin D adequacy in CKD patients would be achieved at a much higher serum concentration of 25-hydroxyvitamin D, since determination of vitamin D adequacy in CKD patients can be much less than 20. Optionally, the ratio of 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D in the patient's serum is lowered or maintained at less than 20, less than 15, or less than 10, for example, the ratio of 25hydroxyvitamin D to 24,25-dihydroxyvitamin D is about 25 , about 24, about 23, about 22, about 21, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6 or example but 5. Quantitative determination of 25-hydroxyvitamin D and 24,25dihydroxyvitamin D can be performed using a suitable assay. Suitable assays are known in the art, eg radioimmunoassay, mass spectrometry, HPLC and LCMS/MS, eg as described in Kaufmann et al., supra, Wagner et al., supra and Berg et al., supra.

Administration of a vitamin D hormone replacement therapy compound alone can decrease serum 25-hydroxyvitamin D and simultaneously increase catabolism by CYP24A, leading to an increase in serum 24,25-dihydroxyvitamin D and a decrease in the ratio of 25-hydroxyvitamin D to 24.25 serum α-dihydroxyvitamin D, but without achieving vitamin D sufficiency. It is possible that administration of a vitamin D hormone replacement therapy compound alone may result in a serum 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D ratio of 20 or less. Accordingly, it is envisaged that in one category of embodiments of the methods described herein, patients are to be treated and will be treated to control the ratio of 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D in a patient at a level of less than 20, and simultaneously to achieve a level Serum 25 hydroxyvitamin D at least 30 ng/mL. In another category of embodiments of the methods described herein, patients will be treated to control the patient's 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D ratio to less than 20, and these patients will be on vitamin D supplementation therapy. Optionally, patients may be treated without administering a vitamin D hormone replacement therapy compound. In one type of embodiment, a method of treating secondary hyperparathyroidism and vitamin D deficiency in a patient with chronic kidney disease comprises administering to said patient repeated doses of a vitamin D supplementation therapy compound effective to increase the level of 25hydroxyvitamin D in the patient's serum to values greater than 90 ng/ml and to control the ratio of 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D in the serum of the subject to values less than 20, and said patient is not taking vitamin D hormone replacement therapy. In another type embodiments, a method for treating secondary hyperparathyroidism and vitamin D deficiency in a patient with chronic kidney disease comprises co-administering repeated doses of a vitamin D supplementation therapy compound and a vitamin D hormone replacement therapy compound to said patient, effect iv to increase the level of 25-hydroxyvitamin D in the patient's serum to values greater than 30 ng/ml and to control the ratio of 25-hydroxyvitamin D to 24,25 dihydroxyvitamin D in the subject's serum to values less than 20.

In another aspect, the present disclosure provides a method of treating vitamin D deficiency in a patient with CKD, comprising administering to said patient repeated doses of 25-hydroxyvitamin D effective to increase the subject's serum 1,25-dihydroxyvitamin D level to values greater than about 40 pg/mL, optionally also to control the ratio of 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D in the patient's serum to values less than 20. Optionally, the concentration of 1,25-dihydroxyvitamin D in the patient's serum is increased to a level of more than about 40, more than 45, more than 50, over 55, over 60, over 65, over 70, over 75, over 80, over 90, over 100, over 110, over 120, over 130, over 140, over 150, over 160, over 170, over 180, over 190 , more than 200, more than 210, more than 220, more than 230, more than 240, more than 250, more than 260, more than 270, more than 280, more than 290 or more than 300 pg / ml. Optionally, the level of 1,25-dihydroxyvitamin D in the patient's serum is increased, for example, to a value of 150 to 350, or 150 to 300 pg/ml, or 200 to 300 pg/ml. Optionally, the patient's serum 1,25-dihydroxyvitamin D concentration is increased from baseline by at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175 or at least 200 pg/ml.

In any of the methods of the present invention, repeated doses of 25-hydroxyvitamin D are optionally administered to the subject in an amount effective to produce one or more effects, including (a) a decrease in PTH, (b) an increase in serum 25-hydroxyvitamin D, (c) an increase in 1, 25

- 9 041576 dihydroxyvitamin D in serum or (d) controlling the ratio of 25-hydroxyvitamin D to 24,25dihydroxyvitamin D, as described herein. Dosing can also be done to avoid one or more negative effects, including (a) no increase in serum calcium, (b) no increase in serum phosphorus, (c) no increase in a marker of bone turnover, (d) no occurrence of hypercalcemia, or ( e) no occurrence of hyperphosphatemia. In one aspect, repeated doses of 25-hydroxyvitamin D are optionally administered to the subject in an amount effective to reduce the patient's blood PTH level (e.g., intact plasma PTH) by at least about 15%, such as at least about 15, at least about 20 is at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, or at least about 50% of its pre-treatment level. In another aspect, repeated doses of 25-hydroxyvitamin D are optionally administered to a patient group in an amount effective to reduce the mean intact PTH plasma level in the patient group by at least about 15%, such as at least about 15, at least about 20, at least at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% of its pre-treatment level.

In one aspect, repeated doses of 25-hydroxyvitamin D are administered in an amount effective to maintain serum calcium at a concentration of about 9.0 to about 10.0 mg/dL, such as about 9.0 to about 9.5 mg/dL, about 9.2 to about 9.6 mg/dl; or about 9.3 to about 9.8 mg/dl. In another aspect, repeated doses of 25-hydroxyvitamin D are administered in an amount effective to increase serum calcium levels from 0 to about 0.3 mg/dl. In one aspect, repeated doses of 25-hydroxyvitamin D are administered in an amount effective to maintain serum phosphorus at about 3.5 to about 4.5 mg/dL, such as about 3.5 to about 4.0 mg/dL, about 3.6 to about 4.2 mg/dl, or about 3.7 to about 4.1 mg/dl. In another aspect, repeated doses of 25-hydroxyvitamin D are administered in an amount effective to increase serum phosphorus by 0 to about 0.3 mg/dl. In one aspect, repeated doses of 25-hydroxyvitamin D are administered in an amount effective to maintain or decrease a bone turnover marker selected from urinary Ca/Cr, urinary TRP, serum BSAP, serum CTX-1, serum P1NP, and their combinations.

In any of the methods described herein, 25-hydroxyvitamin D is optionally administered in modified release formulations, for example, in a sustained release oral dosage form containing 25-hydroxyvitamin D3. In one type of embodiment, said modified release compositions for oral administration of the present invention are designed to contain a concentration of 25-hydroxyvitamin D (e.g., 25-hydroxyvitamin D3 or a combination of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3) from 1 to 1000 , or 1 to 500, or 1 to 100, or 1 to 50, or 10 to 40 micrograms per dose, e.g. 30, 60, 90, 150, 300, 450, 600, 750 or 900 micrograms per dose , and are formulated to provide a controlled or substantially constant release of 25hydroxyvitamin D into the subject's gastrointestinal tract over an extended period of time. In one embodiment, the 25-hydroxyvitamin D is 25-hydroxyvitamin D3. In another embodiment, 25-hydroxyvitamin D is a combination of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 and is suitable for supporting the endocrine systems of vitamin D3 and vitamin D2. Current oral vitamin D supplements, as well as prior oral formulations of 25-hydroxyvitamin D3, support only one or the other of these systems. In one type of embodiment, release may occur in the ileum or later, such as in the colon. In another type of embodiments, the composition may provide substantially improved absorption of 25-hydroxyvitamin D via DBP transport and reduced absorption via chylomicron transport. Examples of modified release formulations of 25-hydroxyvitamin D are described in US Pat. Nos. 8,207,149; 8361488; 8426391; 8778373 and 8906410; as well as in the application for US patent No. 14/213285, the contents of which are incorporated herein by reference.

In one type of embodiments, 25-hydroxyvitamin D is administered orally. For example, 25hydroxyvitamin D can be administered in a modified release oral formulation. Alternatively, 25-hydroxyvitamin D can be administered in an immediate release oral formulation in multiple daily doses to achieve a serum pharmacokinetic profile of 25-hydroxyvitamin D similar to that achieved with a modified or sustained release oral formulation.

The preparation of a modified release dosage form of 25-hydroxyvitamin D suitable for oral administration can be accomplished in many different ways. For example, one or more 25-hydroxyvitamin D compounds can be dispersed in a matrix, ie. in a mixture of components and speed-adjusting agents in carefully selected ratios in the matrix, and optionally coated or encased in a coating material. In a different

- 10 041576 Alternatively, various coating techniques can be used to control the rate and/or site of release of 25-hydroxyvitamin D from the pharmaceutical dosage form. For example, dissolution of the coating may be triggered by the pH of the environment, and subsequent gradual dissolution of the coating over time exposes the matrix, exposing it to fluid from the local environment. In one type of implementation, after the coating becomes permeable, 25-hydroxyvitamin D diffuses from the outer surface of the matrix. When said surface becomes depleted or depleted in 25-hydroxyvitamin D, the underlying reserves begin to be consumed by diffusion through the decomposing matrix into the external solution. In another type of embodiments, the release of 25-hydroxyvitamin D is due to the gradual degradation or erosion of the matrix, for example, due to the solubility of one or more components of the matrix and/or as a result of loss of physical integrity.

In one aspect, the dosage form according to the present disclosure provides one or more 25-hydroxyvitamin D compounds in a matrix that binds the ingredients for sustained release with the possibility of subsequent separation, for example, under the action of the contents of the ileum and/or colon.

Optionally, the dosage form or matrix containing 25-hydroxyvitamin D may have a coating that is resistant to degradation in gastric juice. The modified release dosage form of 25hydroxyvitamin D containing the coating is then orally administered to a subject, eg, to animal or human patients. As the dosage form passes through the proximal small intestine, the enteric coating gradually becomes more permeable, but in an appropriate embodiment, it provides a residual structural framework around the matrix containing 25-hydroxyvitamin D. The matrix containing 25-hydroxyvitamin D becomes significantly more susceptible to intestinal fluids in the ileum through a permeable outer coating, and then 25-hydroxyvitamin D is gradually released through simple diffusion and/or slow degradation of the matrix.

After being released into the ileum, 25-hydroxyvitamin D is absorbed into the lymphatic system or into the portal circulation, where it binds to and is transported by DBP. In a delayed-release embodiment, 25-hydroxyvitamin D is absorbed primarily in a post-duodenal and jejunal position. These proximal parts of the small intestine can respond to high intraluminal concentrations of 25-hydroxyvitamin D and in doing so can catabolize significant amounts of 25-hydroxyvitamin D. By substantially delaying the release of 25-hydroxyvitamin D to the ileum and/or colon, the pharmaceutical composition described herein , can virtually eliminate this possible first pass effect in the proximal intestine and reduce unwanted catabolism. Significant catabolism of the administered 25-hydroxyvitamin D prior to its absorption into the bloodstream significantly reduces its bioavailability. Elimination of the first pass effect reduces the risk of vitamin D toxicity. Significantly delayed release of 25-hydroxyvitamin D (i.e. behind the duodenum and jejunum) markedly reduces the amount of 25-hydroxyvitamin D entering and absorbed from the small intestine via chylomicrons (since formation and absorption chylomicrons occurs primarily in the jejunum) and consequently increases the amount of 25-hydroxyvitamin D absorbed directly through the intestinal wall and on DBP circulating in the lymph or portal circulation.

In one embodiment, a controlled release oral dosage form of 25-hydroxyvitamin D is prepared generally according to the following method. Sufficient 25-hydroxyvitamin D is completely dissolved in a minimum volume of USP grade absolute ethanol (or other suitable solvent) and mixed with appropriate amounts and types of pharmaceutical grade excipients to form a matrix that is solid or semi-solid at room temperature. temperature and normal human body temperature. Such a matrix is completely or almost completely resistant to degradation in the stomach and upper small intestine, and is progressively degraded in the lower small intestine and/or colon.

In a suitable dosage form, the matrix binds the 25-hydroxyvitamin D compound(s) and allows slow, relatively sustained, e.g., substantially constant release of 25-hydroxyvitamin D over 4 to 8 hours or more, via simple diffusion and/or gradual degradation into the contents of the cavity of the lower small intestine and/or large intestine. The dosage form optionally further comprises an enteric coating that is partially soluble in aqueous solutions at a pH of about 7.0-8.0, or simply dissolves slowly enough such that significant release of 25-hydroxyvitamin D occurs later, after passage of the dosage form through the duodenum and jejunum.

The means for providing controlled release of 25-hydroxyvitamin D may be selected from any suitable controlled release delivery system, including any known active ingredient controlled release delivery system, including the wax matrix system and the EUDRAGIT RS/RL system (Rohm Pharma, GmbH, Weiterstadt, Germany).

The wax matrix system provides a lipophilic matrix. In the wax matrix system, for example, natural or synthetic waxes, digestible or non-digestible waxes, for example beeswax, white wax, sperm whale wax or similar compositions or mixtures of waxes can be used. In one type of embodiments, the wax is a non-digestible wax. The active ingredient(s) are dispersed in a waxy binder which is slowly broken down in the intestinal fluids, gradually releasing the active ingredient(s). The wax binder impregnated with 25-hydroxyvitamin D can be filled into capsules, such as soft gelatin capsules. The soft gelatin capsule may contain one or more gelling agents, for example gelatin, starch, carrageenan and/or other pharmaceutically acceptable polymers. In one embodiment, partially cross-linked gelatin soft capsules are used. Alternatively, vegetable-based capsules may be used. The wax matrix system provides dispersion of the active ingredient(s) in a wax binder that softens at body temperature and slowly disintegrates in the intestinal fluids, gradually releasing the active ingredient(s). In particular, the system may contain a mixture of waxes with the optional addition of oils to achieve a melting point above body temperature, but below the melting point of selected compositions used to obtain a soft or hard capsule shell, or a vegetable-based capsule shell, or other composition used to obtain capsule shell or other coating.

In one type of embodiment, the wax matrix contains a controlled release agent, an emulsifier, and an absorption enhancer. Examples of controlled release agents suitable for use include, but are not limited to, waxes, including synthetic waxes, microcrystalline wax, paraffin wax, carnauba wax, and beeswax; polyethoxylated castor oil derivatives, hydrogenated vegetable oils, glyceryl mono-, di- or tribehenates; long chain alcohols such as stearyl alcohol, cetyl alcohol and polyethylene glycol; and mixtures of any of the above compounds. In particular, indigestible waxy substances are provided, for example hard paraffin wax. The controlled release agent may be present in an amount of at least 5 or more than about 5% by weight of the dosage form. For example, depending on the controlled release agent used, the controlled release agent may be at least 5, or at least 10, or at least 15, or at least 20, or at least 25, or more than 5, or more than 10, or more than 15, or more than 20, and/or more than 25 wt.% of the dosage form. The controlled release agent may be present in an amount of 50%, 40%, 35%, or 30% by weight or less. Suitable ranges include 5 to 40% by weight, 10 to 30% by weight, and 15 to 25% by weight. Examples include about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, and about 25 wt. %.

Examples of emulsifiers suitable for use in dosage form include, but are not limited to, lipophilic agents having an HLB of less than 7, such as mixed fatty acid monoglycerides, mixed fatty acid diglycerides; mixtures of mono- and diglycerides of fatty acids; lipophilic esters of polyglycerol; esters of glycerol, including glyceryl monooleate, glyceryl dioleate, glyceryl monostearate, glyceryl distearate, glyceryl monopalmitate, and glyceryl dipalmitate; glyceryl-lactoesters of fatty acids; esters of propylene glycol, including propylene glycol monopalmitate, propylene glycol monostearate and propylene glycol monooleate; esters of sorbitol, including sorbitan monostearate, sorbitan sesquioleate; fatty acids and their soaps, including stearic acid, palmitic acid and oleic acid, and mixtures thereof; glyceryl monooleate, glyceryl dioleate, glyceryl monostearate, glyceryl distearate, glyceryl monopalmitate and glyceryl dipalmitate; glyceryl-lactoesters of fatty acids; esters of propylene glycol, including propylene glycol monopalmitate, propylene glycol monostearate and propylene glycol monooleate; esters of sorbitol, including sorbitan monostearate, sorbitan sesquioleate; fatty acids and their soaps, including stearic acid, palmitic acid and oleic acid; and their mixtures.

One type of lipophilic agents is selected from glycerides and their derivatives. The glycerides are selected from the group consisting of, for example, medium or long chain glycerides, caprylocaproyl macrogol glycerides, and mixtures thereof. Medium chain glycerides may include, but are not limited to, medium chain monoglycerides, medium chain diglycerides, caprylic/capric triglyceride, glyceryl monolaurate, glyceryl monostearate, caprylic/capric glycerides, glyceryl monocaprylate, glyceryl monocaprylate, caprylic/capric/linoleic triglyceride, and caprylic/capric/capric triglyceride. In particular, low melting point monoglycerides are provided for the preparation of dosage forms. Monoglycerides may include, but are not limited to, glyceryl monostearate, glyceryl monopalmitate, glyceryl monooleate, glyceryl monocaprylate, glyceryl monocaprate, glyceryl monolaurate, and the like. In particular, glycerol monostearate (GMS) is provided. GMS is a natural emulsifying agent. It is soluble in oil, but slightly soluble in water. GMS has a GBL value of 3.8. The lipophilic emulsifier may be present in an amount of from about 10% to about 40% by weight or, for example, from about 20% to

- 12 041576 about 25 wt.%. Other examples include about 20%, about 21%, about 22%, about 23%, about 24%, and about 25% by weight.

Examples of suitable absorption enhancers include, but are not limited to, caprylocaproyl macrogol triglycerides such as polyethylene glycosylated glycerides, also known as polyglycosylated glycerides or PEGylated glycerides. PEGylated glycerides that can be used in the present composition include, but are not limited to, mixtures of monoglycerides, diglycerides and triglycerides and polyethylene glycol monoesters and diesters, polyethylene glycosylated almond glycerides, polyethylene glycosylated corn glycerides, and polyethylene glycosylated caprylic/capric triglycerides. The absorption enhancer may have an HLB value of 13 to 18 or 13 to 15. One class of absorption enhancer is known under the trade name GELUCIRE (Gattefosse Corporation, Paramus, NJ, USA). GELUCIRE is a well-known excipient that is a family of fatty acid esters with glycerol and PEG esters, also known as polyglycosylated glycerides. GELUCIRE is used in a variety of applications including the preparation of sustained release pharmaceutical compositions. GELUCIRE compounds are inert, semi-solid waxy substances that are amphiphilic and are available in various physical properties such as melting point, HLB and solubility in various solvents. They are surface active in nature and disperse or solubilize in an aqueous medium to form micelles, microscopic globules or vesicles. They are distinguished by their melting point/HLB value. The melting point is expressed in degrees Celsius. One or a mixture of different grades of GELUCIRE excipients can be selected to achieve desired melting point and/or HLB characteristics. In particular, GELUCIRE 44/14 (lauroyl macrogol glycerides/lauroyl polyoxyl glycerides) is provided which has a melting point of 44°C and a HLB of 14. The absorption enhancer may be present in an amount of from about 5 to about 20 wt.% or, for example, from about 8 to about 15 wt.%. Other examples include about 8, about 9, about 10, about 11, about 12, about 13, about 14, and about 15% by weight.

Examples of other lipid matrices suitable for use in the methods of the present invention include one or more of glycerides, fatty acids and alcohols, and fatty acid esters.

In one embodiment, the dosage form may contain an oily carrier for the 25-hydroxyvitamin D compound. Any pharmaceutically acceptable oil may be used. Examples include animal (eg, fish), vegetable (eg, soy) and mineral oils. The oil may be chosen to readily dissolve the 25hydroxyvitamin D compound used. Oil carriers may include indigestible oils such as mineral oils, in particular liquid paraffins, and squalene. The ratio between wax matrix and carrier oil can be optimized to achieve the desired release rate of the 25hydroxyvitamin D compound. Thus, if a heavier oil component is used, relatively less wax matrix can be used, and if a lighter oil component is used, relatively more wax matrix can be used. . In one embodiment, the particular choice of oil carrier provides controlled release so that absorption of 25-hydroxyvitamin D occurs later when the dosage form reaches the jejunum and/or colon.

In one embodiment, the dosage form may contain a stabilizing agent such as a cellulose compound. Examples of cellulose compounds and stabilizing agents include, but are not limited to, uronic acid cellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose (hypromellose), methylcellulose, polyanionic cellulose, and combinations thereof. Also provided are one or more poloxamers (eg, poloxamer 407), poly(ethylene oxide) polymers (eg, Dow's POLYOX polymers), povidone, and fumed silica (eg, AEROSIL 200, Evonik Industries AG, Essen, Germany). A stabilizing agent, such as a cellulose compound, may be present, for example, in an amount of at least about 5% of the dosage form relative to the total weight of the dosage form, excluding any additional coatings or shells (wt.%). For example, the cellulosic compound may be present in an amount of at least 5%, or at least 10%, or at least 15% by weight of the dosage form, or more than 5%, or more than 10%, or more than 15% by weight of the dosage form. Suitable ranges include 5 to 30 wt%, 10 to 20 wt%, 10 to 15 wt%, 5 to 15 wt%, and 7.5 to 12.5 wt%. Examples include about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, and about 15 wt%. It should be understood that a stabilizing agent referred to in this context is an agent that stabilizes the release profile upon dissolution (and therefore also the in vivo release profile) against significant change over time under storage conditions, e.g. under normal warehouse storage conditions. . other agents,

- 13 041576 which are known in the art as preservatives to prevent degradation of the active agent itself, are not included in the meaning of the terms stabilizing agent and stabilizer, although such preservatives are also provided for use in dosage forms according to the present description.

In particular, in one suitable type of implementation, the waxes selected for the matrix are melted and thoroughly mixed, optionally at a temperature of about 80°C. The required amount of oil(s), lauroyl-polyoxylglycerides, mono- and diglycerides, butylhydroxytoluene, and hypromellose are then added, followed by sufficient mixing to homogenize. Then the wax mixture is gradually cooled to a temperature just above its melting point, optionally to a temperature of from about 50 to about 62°C. The mixing steps are optionally carried out under vacuum. The required amount of 25-hydroxyvitamin D dissolved in ethanol is evenly distributed in the molten matrix, optionally under vacuum and/or at a temperature of from about 59 to about 62°C. The matrix is loaded into capsules, such as vegetable-based or gelatin-based capsules, optionally under vacuum and/or at a temperature of from about 63 to about 70°C.

Filled capsules are optionally treated for a suitable period of time with a solution containing an aldehyde, such as acetaldehyde, to partially cross-link the polymer, eg gelatin, in the capsule shell, if used. Within a few weeks, the degree of cross-linking of the capsule shell increases and thus it becomes more resistant to dissolution in the contents of the stomach and upper intestine. When properly formulated, such a gelatinous shell gradually dissolves after oral administration and becomes sufficiently porous (without complete decomposition) by the time it reaches the jejunum, allowing slow diffusion of 25-hydroxyvitamin D from the waxy matrix into the contents of the lower small intestine and/or large intestine.

Another suitable controlled release oral drug delivery system is the EUDRAGIT RL/RS system in which the 25hydroxyvitamin D active ingredient is formulated into 25/30 mesh granules. The granules are then uniformly coated with a thin coating of polymer lacquer, which is insoluble in water but slowly permeated by water. The coated granules may be mixed with optional additives including one or more of antioxidants, stabilizers, binders, lubricants, processing aids, and the like. The resulting mixture can be compressed into a tablet which is hard and dry before use and can be further coated or encapsulated. After swallowing a tablet or capsule and bringing it into contact with aqueous intestinal fluids, a thin layer of varnish begins to swell and allows the intestinal fluids to slowly penetrate. As the intestinal fluid slowly penetrates the varnish coating, 25-hydroxyvitamin D is slowly released. By the time the tablet or capsule has passed through the small intestine, after about 4-8 hours or more, there is a slow but complete release of 25-hydroxyvitamin D. Accordingly, the swallowed tablet releases a flood of 25-hydroxyvitamin D as well as any other active ingredient.

The EUDRAGIT system consists of high permeability lacquers (RL) and low permeation lacquers (RS). RS is a water-insoluble film former based on neutral, swellable esters of methacrylic acids, with a small content of trimethylammonioethyl methacrylate chlorides; the molar ratio of quaternary ammonium groups to neutral ester group is about 1:40. RL is also a water-insoluble, swellable film former based on neutral methacrylic acid esters with a small content of trimethylammonioethyl methacrylate chloride, the molar ratio of quaternary ammonium groups to neutral ester groups is about 1:20. The permeability of the coating and hence the duration of drug release can be adjusted by changing the ratio of RS to RL in the coating material. Additional details regarding the Eudragit RL/RS system are available in technical publications from Rohm Tech, Inc. 195 Canal Street, Malden, MA 02146, and Lehmann et al., Int. J Pharm. Tech. & Prod. mfr. 2(r), 31-43, 1981, the contents of which are incorporated herein by reference.

Other examples of insoluble polymers include polyvinyl esters, polyvinyl acetals, polyacrylic acid esters, styrene-butadiene copolymers, and the like.

In one embodiment, after the coated beads have been formed into a tablet or encapsulated, the tablet or capsule is coated with an enteric material that dissolves at a pH of 7.0 to 8.0. One such pH dependent enteric coating material is EUDRAGIT L/S, which is soluble in intestinal fluid but not in gastric juice. Other enteric coating materials can be used, such as cellulose acetate phthalate (CAP), which is resistant to dissolution by the action of gastric juice, but is easily degraded due to the hydrolytic effect of intestinal esterases.

In one embodiment, the particular choice of enteric coating material and controlled release coating material provides controlled and substantially constant release over 4-8 hours or more such that significant release occurs later when the dosage form reaches the jejunum. Optionally, a controlled release formulation of the present disclosure administered once daily may specifically provide substantially constant intraluminal, intracellular, and circulatory levels of 25hydroxyvitamin D when compared to an equal dose of an immediate release 25hydroxyvitamin D formulation administered alone. once a day.

Dosage forms may also contain adjuvants such as preservative adjuvants. Dosage forms according to the present invention may also contain other therapeutically important substances, or may contain more than one compound specified in this document and in the claims, in the form of a mixture.

As an alternative to oral 25-hydroxyvitamin D, intravenous administration of 25-hydroxyvitamin D is also contemplated. In one embodiment, 25-hydroxyvitamin D is administered as a sterile intravenous injection, optionally a bolus injection, of a composition that provides a sustained release profile. In another embodiment, the 25hydroxyvitamin D is administered by gradual injection/infusion, eg over 1 to 5 hours, to provide a controlled or substantially constant release of the 25hydroxyvitamin D directly into the DBP in the patient's blood. For example, the composition can be administered by injection or infusion over at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, or, for example, at least about 6 hours. and up to 24 hours. In one embodiment, the composition intended for intravenous administration according to the present description, is designed to contain a concentration of compound(s) of 25-hydroxyvitamin D from 1 to 100 μg per dose. Sterile, isotonic dosage forms of 25-hydroxyvitamin D can be prepared by dissolving 25-hydroxyvitamin D in absolute ethanol, propylene glycol or other suitable solvent and combining the resulting solution with one or more surfactants, salts and preservatives in appropriate volumes of water for injection. Such dosage forms can be administered slowly from syringes, for example, through heparin locks, or by adding a larger volume to sterile solutions (eg, saline), with a steady infusion over a certain time.

A particular dosage form of 25-hydroxyvitamin D ₃ for use in the methods of the present disclosure is a sustained release oral dosage form containing 25-hydroxyvitamin D (e.g., about 30, about 60, about 90, about 150, about 300, about 450, about 600, about 750 or about 900 μg of 25-hydroxyvitamin D ₃ ), about 2 wt.% (for example, 2.32 wt.%) anhydrous/anhydrous ethanol, about 10 wt.% (for example, 9.75 wt. .%) lauroyl-polyoxylglycerides (for example, GELUCIRE 44/14), about 20 wt.% (for example, 20.00 wt.%) solid paraffin, about 23 wt.% (for example, 22.56 wt.%) mono- and diglycerides (e.g. GMS), about 35 wt.% (e.g. 35.34 wt.%) liquid paraffin or mineral oil, about 10 wt.% hypromellose and optionally a small amount of preservative (for example, 0.02 wt.% BHT ). The dosage form is optionally enclosed in a soft capsule shell, e.g., a capsule shell containing modified starch, carrageenan (e.g., iota and/or kappa), dibasic sodium phosphate, sorbitan sorbitan solution, color (e.g. drugs and cosmetics), titanium dioxide and purified water. In one aspect, the 90% confidence interval (90% CI) of the ratio of the mean Cmax between the experimental dosage form and the dosage form according to the present description is from 80 to 125% (0.8-1.25). In another aspect, the 90% confidence interval (90% CI) of the ratio of mean AUC( _0-in f) between the experimental dosage form and the dosage form according to the present description is from 80 to 125% (0.8-1.25). Optionally, the 90% confidence interval (90% CI) of the Tmax relationship between the experimental dosage form and the dosage form according to the present description is from 80 to 125% (0.81.25). A dosage form that is bioequivalent to the dosage form described herein is also specifically contemplated for use in the methods of the present disclosure.

In one aspect, the present disclosure provides a method of treating secondary hyperparathyroidism in a patient with stage 3 or 4 chronic kidney disease and a serum total 25-hydroxyvitamin D level of less than 30 ng/mL, comprising administering an oral sustained release formulation of calcifediol. Optionally, the patient's serum calcium level is less than 9.8 mg/dl and/or the patient's serum phosphorus level is less than 5.5 mg/dl. Optionally, said method comprises administering a sustained release oral formulation of 25-hydroxyvitamin D ₃ containing 30 μg of calcifediol once daily, eg, at bedtime. In one embodiment, said method comprises administering 60 μg of calcifediol to increase serum total 25-hydroxyvitamin D to the range of 30 to 100 ng/mL and to decrease intact plasma parathyroid hormone (iPTH) to the iPTH value that is the target of treatment. , provided that the content of calcium

- 15 041576 serum level is within the normal reference range. In various embodiments, said method comprises administering from about 30 to about 60 micrograms of calcifediol per day. In some embodiments, said method comprises administering 25-hydroxyvitamin D in an amount of from about 300 to about 900 micrograms per week, such as 600 micrograms per week, optionally in divided doses of two or three doses per week, such as three times per week at dialysis treatment.

In one embodiment, calcifediol is synthetically produced in the form of calcifediol monohydrate. Calcifediol monohydrate is a white crystalline powder with an estimated molecular weight of 418.65, soluble in alcohol and fatty oils, but practically insoluble in water. From a chemical point of view, calcifediol monohydrate is (3p,5Z,7E)-9,10secocholesta-5,7,10(19)-triene-3,25-diol monohydrate and has the following structural formula:

Further, the present invention is explained in the following examples, which should in no way be construed as limiting the scope of the present invention.

Example 1 Phase III clinical trial of extended release 25-hydroxyvitamin D ₃ in patients with stage 3 or 4 CKD.

The efficacy and safety of the extended release formulation of 25-hydroxyvitamin D ₃ (RAYALDEE, Opko Ireland Global Holdings Ltd.) was evaluated in two identical multicenter, randomized, placebo-controlled, double-blind studies in patients with secondary hyperparathyroidism (iPTH >85 pg/ ml), stage 3 or 4 chronic kidney disease, and associated serum levels of total 25-hydroxyvitamin D >10 and <30 ng/mL. Subjects were divided into disease stages and randomized in a 2:1 ratio to a single daily dose of 30 μg oral dose of RAYALDEE (or matching placebo) at bedtime for 12 weeks, followed by an additional 14 weeks of treatment with a single daily dose of 30 or 60 mcg oral RAYALDEE (or placebo) at bedtime. Each capsule contained the following excipients: mineral oil, monoglycerides and diglycerides, paraffin, hypromellose, lauroyl polyoxyl glycerides, anhydrous alcohol and butylhydroxytoluene. The capsule shells contained modified starch, carrageenan, dibasic sodium phosphate, sorbitan sorbitan solution, food, pharmaceutical, and cosmetic blue #1, titanium dioxide, and purified water. Medium chain triglyceride (fractionated coconut oil) was used as a lubricant in the manufacturing process and trace amounts may be present in the finished dosage form.

Overall, 213 subjects were randomized for one study (72 to placebo and 141 to RAYALDEE), and 216 subjects were randomized to the other study (72 to placebo and 144 to RAYALDEE). The mean age of the subjects was 66 years (range 25 to 85), 50% were male, 65% were white, 32% were African American or black, and 3% were other race. At entry into the study, subjects were diagnosed with secondary hyperparathyroidism, stage 3 (52%) or stage 4 (48%) CKD without macroalbuminuria, and serum total 25hydroxysitamine D was less than 30 ng/mL. The most common cause of CKD was diabetes and hypertension, and the mean estimated glomerular filtration rate (GFR) at baseline was 31 ml/min/1.73 m ² . At study entry, mean intact serum PTH was 148 pg/ml, mean serum calcium was 9.2 mg/dl, mean serum phosphorus was 3.7 mg/dl, and mean 25-hydroxyvitamin D in serum was 20 ng/ml. Mean baseline iPTH was 130 pg/mL for subjects with stage 3 disease (n=122) and 166 pg/mL for subjects with stage 4 disease (n=207). Of the 429 subjects, 354 (83%) completed the study and 298 (69%) were selected to continue treatment for an additional 6 months with RAYALDEE in an open label extension study. All subjects started at a dose of 30 mcg taken once a day at bedtime, and most (210, 74%) increased the dose to 60 mcg after 12 weeks if the content of intact plasma PTH was more than 70 pg/ml, the content of 25- serum hydroxyvitamin D was less than 65 ng/ml and serum calcium was less than 9.8 mg/dl.

The primary analysis determined the proportion of individuals who had at least a 30% reduction in intact plasma PTH from baseline at the end of the study (average of weeks 20, 22, 24, and 26). In both trials, more patients randomized to RAYALDEE showed at least a 30% reduction in intact plasma PTH from baseline compared with placebo (33% versus 8% in the first trial).

- 16 041576 (P<0.001) and 34% vs. 7% in the second trial (P<0.001)).

Mean plasma iPTH gradually decreased to nearly normal levels during 1 year of RAYALDEE treatment regardless of CKD stage, but increased in the placebo treatment group. At the end of 26 weeks of treatment, pooled plasma mean iPTH decreased by 22±32% with RAYALDEE treatment but increased by 9±36% with placebo treatment (Fig. 1A), with comparable efficacy in patients with CKD 3 and 4 stages (Fig. 1B). The suppression of iPTH was directly proportional to the increase in total serum 25hydroxyvitamin D (Fig. 1C) and was constant over the 6-month follow-up study.

Serum total 25-hydroxyvitamin D increased gradually to >30 ng/mL in 80% and 83% of RAYALDEE-treated subjects compared to 3% and 7% of placebo-treated subjects (p<0.001) in these two studies. Mean steady-state levels were 50 ± 20 and 56 ± 19 ng/mL for subjects taking 30 mcg per day, and 69 ± 22 and 67 ± 21 ng/mL for subjects taking 60 mcg per day, in the first and second studies, respectively ( Fig. 2A). Serum 25hydroxyvitamin D levels of even about 185 ng/ml have been achieved without any adverse reactions. These results were comparable to a previous repeat dose clinical study in which 30 µg daily doses of RAYALDEE provided a gradual increase in mean serum 25-hydroxyvitamin D to a total of 5.8 ± 1.2 (CO) ng/mL/week, and the corresponding the increase in mean serum 1,25-dihydroxyvitamin D was, in general, 6.7 ± 3.1 pg/ml/week. During the first two weeks of treatment with RAYALDEE, a decrease in circulating intact plasma PTH was observed.

The response rates for plasma iPTH and total serum 25-hydroxyvitamin D were similar in patients with stage 3 and 4 CKD, as was the increase in total serum 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D. Mean serum total 1,25D increased significantly at the end of the treatment period, regardless of the stage of CKD (FIG. 2B).

Of the 285 subjects treated with RAYALDEE, 179 showed baseline levels of 24.25(OH) ₂ D ₃ that were above the lower limit of quantitation (LOQ) (<0.51 ng/mL). During 26 weeks of treatment with RAYALDEE, mean 25(OH)D3 and 24.25(OH)2D3 increased significantly (p<0.0001 vs. placebo) from baseline, from 29 to 87 ng/mL and from 1.0 to 4.1 ng/ml (Fig. 3A), respectively; mean iPTH decreased significantly (p<0.0001) from 147 to 111 pg/mL compared to the mean increase in placebo subjects. The 25(OH)D3:24.25(OH)2D3 ratios were variable (OCO ~30%) but decreased with RAYALDEE treatment from a mean of 30 to 25 (Figure 3B) and were significantly lower at 26 weeks. than in the placebo group (p<0.0001). These results demonstrate that the ratio 25(OH)D ₃ :24.25(OH) ₂ D ₃ can be used to diagnose vitamin D deficiency (VDI) in patients with CKD. The data further suggest that reducing this ratio to <20 requires a 25(OH)D ₃ level of more than 85 ng/mL and that the 25(OH)D target in CKD patients is higher than would be expected for healthy subjects.

No differences in urinary calcium or phosphorus were observed between treatment groups. Mean serum Ca and P levels of subjects treated with extended release calcifediol were broadly similar to those of subjects in the placebo treatment group (FIG. 6A); at some points in time, slight increases were noted. Mean±CO serum calcium adjusted for albumin increased in pooled data from baseline of 9.2±0.3 mg/dl by 0.2±0.3 mg/dl in the RAYALDEE group compared with 0.1±0.3 mg/dl in placebo treatment. Serum phosphorus with RAYALDEE increased by 0.2±0.5 mg/dl from baseline of 3.7±0.6 mg/dl in the RAYALDEE treatment group compared to 0.1±0. 4 mg/dL relative to baseline of 3.8±0.5 when treated with placebo. No relationship was observed between treatment and response in terms of serum calcium (FIG. 6D) or serum phosphorus (FIG. 6C) relative to the amount of 25-hydroxyvitamin D3 administered. Overall, 6 subjects (2%) in the RAYALDEE treatment group required a dose reduction upon reaching the protocol-approved criteria for albumin-adjusted hypercalcemia (two consecutive serum calcium values >10.3 mg/dL), according to compared with no such subjects in the placebo group. Five of these subjects had a history of serum calcium >9.8 mg/dl prior to treatment, and a sixth subject was concurrently on thiazide treatment. Overall, 4.2% of subjects taking RAYALDEE and 2.1% of subjects taking placebo experienced >1 increase in serum calcium above the upper limit of normal (10.5 mg/dL). Adverse events of elevated blood calcium or hypercalcemia occurred in 0.7% of subjects in the RAYALDEE treatment group and 1.4% of subjects in the placebo treatment group. One subject (0.4%) in the RAYALDEE treatment group achieved the approved protocol

- 17 041576 criteria for hyperphosphatemia (two consecutively measured serum phosphorus values >5.5 mg/dl, presumably due to the administration of the experimental drug) and required a dose reduction compared to no such subjects in the placebo group. Overall, 45.6% of subjects taking RAYALDEE and 44.4% of subjects taking placebo experienced >1 increase in serum phosphorus above the upper limit of normal (4.4 mg/dL). Adverse events of increased blood phosphorus or hyperphosphatemia occurred in 1.8% of subjects in the RAYALDEE treatment group and in 2.8% of subjects in the placebo treatment group.

The content of bone tissue renewal markers decreased during treatment with RAYALDEE. Urinary calcium/creatinine ratio (Ca/Cr) and tubular reabsorption of phosphate (TRP) remained unchanged (FIGS. 6D and 6E). Serum total alkaline phosphatase decreased from 93 to 87 U/L, serum bone-specific alkaline phosphatase (BSAP) decreased from 38 to 27 U/L (Fig. 7A), C-terminal telopeptide-1 (CTX- 1) in serum decreased from 734 to 612 pg/ml (Fig. 7B), and the serum amino-terminal propeptide of procollagen-1 (P1NP) decreased from 99 to 89 ng/ml (Fig. 7C). When administered with placebo, these bone markers either increased or remained relatively unchanged.

A study on the effect of a 450 µg supertherapeutic dose meal in healthy subjects showed an approximately 5-fold increase in maximum calcifediol serum concentration (Cmax) and a 3.5-fold increase in AUC _0-t when RAYALDEE was administered with a high-calorie, high-fat meal. compared to its administration on an empty stomach.

A single supertherapeutic dose study of 900 μg conducted in healthy subjects demonstrated peak blood concentrations of calcifediol between 21 and 32 hours post-dose in the fasting state.

Example 2 Pharmacokinetic Studies.

The pharmacokinetics of RAYALDEE was studied in healthy subjects and in subjects with stage 3 or 4 CKD. Exposure to calcifediol increased proportionately over the dose range of 30 to 90 mcg following repeated daily administration of RAYALDEE at bedtime in subjects with secondary hyperparathyroidism, chronic kidney disease, and vitamin D deficiency. Stationary serum levels of total 25hydroxyvitamin D were reached after approximately 3 months. After multiple doses of RAYALDEE, mean steady-state serum total 25-hydroxyvitamin D concentrations were 53 and 68 ng/mL in the 30 and 60 µg dose groups, respectively. In terms of distribution, calcifediol was actively bound to plasma proteins (>98%). The mean apparent volume of distribution was 8.8 L in healthy subjects after a single oral dose of RAYALDEE, and 30.1 L in subjects with stage 3 or 4 chronic kidney disease after repeated doses. In terms of elimination, the mean elimination half-life of calcifediol was approximately 11 days in healthy individuals after a single dose of RAYALDEE, and approximately 25 days in patients with stage 3 or 4 chronic kidney disease after repeated once-daily dosing. In terms of metabolism, the formation of calcitriol from calcifediol is catalyzed by the enzyme 1αhydroxylase, CYP27B1, which is found in the kidney, parathyroid gland, and other tissues. CYP24A1, which is found in all vitamin D-sensitive tissues, catabolizes calcifediol and calcitriol to inactive metabolites. Calcitriol inhibits CYP27B1 and activates CYP24A1. Excretion of calcifediol occurs mainly through the biliary tract and with feces. No dependence of steady-state concentrations of calcifediol on CKD stage was observed after daily administration of RAYALDEE, according to the results of pharmacokinetic analysis in the group of subjects with stage 3 or 4 CKD. In table. 1 summarizes baseline-adjusted pharmacokinetic parameters of calcifediol by treatment group.

_________________________________________________Table 1

Placebo 30 mcg 60 mcg 90 mcg

Initial Value (ng/ml) Average (SD) 16.4 (8.2) 16.2 (7.3) 19.8 (8.7) 18.4 (9.8) Median 12.9 17.2 21.2 16, 7 Minimum, maximum 4.4, 30.4 5.0, 25.8 5.8, 32.5 6.7, 38.9

- 18 041576

Stach (NG/ML)

Average (SD) 4.1 (3.5) 27.8 (8.2) 60.3 (19.0) 85.7 (26.9) Median 3.1 28.1 60.8 76.0 Minimum, maximum 0.6, 13.8 10.8, 43.4 30.3, 89.5 55.4, 146.4 AUCq-6 weeks (ng -d/ml) Average (SD) 45.9 (60.0) 709.2 1531.4 2134.3 (246.3) (374.8) (584.3) Median 32.1 6843.0 1573.0 1963.8 Minimum, maximum -60.1, 222.3 307.8, 712.7, 1377.5, 1249.0 2221.8 3207.3 tmax (D) Average (SD) nd 37.8 (10.4) 41.1 (5.2) 42.6 (5.3) Median nd 42.50 43.0 43.0 Minimum, maximum nd 8.0, 44.0 29.0, 45.0 35.0, 57.0 tl/2 (D) Average (SD) nd 25.8 (16.3) 33.1 (9.3) 50.1 (51.0) Median nd 24.1 31.6 37.7 Minimum, maximum nd 5.2, 52.6 17.4, 52.3 23.2, 224.0

In table. 2 summarizes the pharmacokinetic parameters of 1,25-dihydroxyvitamin D adjusted for baseline by treatment group.

_____________________________________________Table 2

Placebo 30 mcg 60 mcg 90 mcg

N=23 N=12 N=16 №14

Initial Value (pg/ml)

Average (CO) 20.8 (10.11) 18.3 (7.53) 20.6 (7.62) 20.6 (7.29) Median 17.0 17.0 18.0 21.0 Minimum, 7.0, 41.4 5.1, 30.7 8.2, 33.6 9.3, 34.5 maximum Stach (PG/ML) Average (CO) 7.6 (5.71) 6.4 (7.66) 18.4 (6.24) 19.9 (14.30) Median 4.9 5.0 18.4 18.9 Minimum, 1.9, 22.6 -6.3, 21.0 7.3, 29.9 -11.6, 48.3 maximum AUCq-6 weeks (g -d/ml) Average (CO) 11.5 (112.97) 100.6 (185.38) 249.9 (198.83) 371.1 (290.81) Median 16.2 23.0 298.7 352.2 Minimum, maximum -267.1, 219.8 -145.4, 452.3 -191.7, 563.6 -5.8, 1235.8 tmax ( D ) Average (CO) 24.4 (15.55) 16.8 (16.09) 26.4 (11.52) 25.5 (13.88) Median 23.0 12.0 23.0 23.00 Minimum, 2.0, 45.0 1.0, 44.0 8.0, 44.0 1.0, 44.0

maximum

Example 3 Clinical Trial of 25-Hydroxyvitamin D ₃ Extended Release in Patients with Stage 5 CKD.

A multicenter, randomized, double-blind, placebo-controlled clinical trial was conducted to evaluate the safety and efficacy of three weekly doses of extended-release formulation of 25-hydroxyvitamin D ₃ versus placebo in reducing intact parathyroid hormone (iPTH) serum levels by at least less than 30% of pre-treatment baseline in subjects with secondary hyperparathyroidism, vitamin D deficiency, and stage 5 CKD undergoing hemodialysis three times a week. A sustained release formulation of 25-hydroxyvitamin D ₃ was provided as a capsule having the same composition as RAYALDEE but containing 150 μg of 25-hydroxyvitamin D ₃ . Approximately 600 subjects were screened for randomization of approximately 280 eligible subjects balanced for the severity of secondary hyperparathyroidism (SHPT) at a ratio of 1:1:1:1 into four parallel groups receiving the following treatment for 52 weeks: (a) drug 25-hydroxyvitamin D ₃ extended-release formulation at 300 µg per week, (b) 25-hydroxyvitamin D ₃ extended-release formulation at 600 µg per week, (c) 25-hydroxyvitamin D ₃ extended-release formulation at a dose of 900 micrograms per week, or (d) a matching placebo.

Subjects must be at least 18 years of age to participate in the study, and subjects must have been diagnosed with stage 5 CKD requiring inpatient hemodialysis three times a week for the previous 9 months, as confirmed by their medical history. At the screening visit, the subject must demonstrate a serum iPTH level of >150 and <600 pg/mL if taking calcitriol or an active vitamin D analog, or a serum iPTH level of >400 and <900 pg/mL if not taking calcitriol or active vitamin D analog. Subjects taking cinacalcet, etelcalcetide, calcitriol, or other 1-hydroxylated vitamin D analogs or approximately 1000 IU/day vitamin D supplements should discontinue further use of these agents during the course of the study and complete the 4-week washout period before starting the 52-week treatment period to achieve serum iPTH >400 and <900 pg/mL, serum calcium <9.8 mg/dL, and serum total 25-hydroxyvitamin D

- 19 041576 <30 ng/ml.

To assess safety and efficacy, blood samples were taken from all subjects at the start of a mid-week dialysis session at one-week or biweekly intervals during the pre-treatment washout period, during the 52-week treatment period, and during the 6-week observation period after treatment. Subjects enrolled in the study in the 600 or 900 mcg group took one or two capsules containing 150 mcg of 25-hydroxyvitamin D ₃ or placebo three times a week (for example, on Monday, Wednesday and Friday or Tuesday, Thursday and Saturday ) within 52 weeks. Subjects in the 300 μg group took one capsule containing 150 μg of 25-hydroxyvitamin D ₃ twice a week (eg, Monday/Tuesday and Friday/Saturday). The capsules were administered at the dialysis center at the end of scheduled hemodialysis to achieve the cumulative cumulative weekly dose. During the study, subjects were on hemodialysis with dialysate calcium < 2.5 mEq/L. In a subgroup of subjects in each experimental group, additional blood samples were collected during the observation period after treatment to determine the pharmacokinetic profile of 25-hydroxyvitamin D ₃ in serum after the last administered dose.

Subjects met the criteria for a dose change at the start of week 26 if (a) iPTH had not decreased by at least 40% from pre-treatment baseline and was maintained at >300 pg/mL, (b) serum calcium was <9, 8 mg/dl, (c) serum phosphorus was <6.0 mg/dl, and (d) the dose administered in this study was less than 900 mcg per week. Dose changes were made in increments of 300 μg per week unless smaller increments were needed to prevent exceeding the maximum dose of 900 μg per week. Subjects were dose reduced by 150 µg per week if iPTH was found to be <150 pg/mL, if serum calcium was found to be >10.3 mg/dL, or if phosphorus was found to be serum phosphorus is >6.5 mg/dL, provided that the investigator associated the increased serum phosphorus with the administration of the experimental drug and took appropriate and ongoing action to control serum phosphorus by initiating or changing treatment with phosphate binders. The patient dose was left unchanged if iPTH was found to be <100 pg/mL or serum calcium was found to be >11.0 mg/dL, and returned to the original dose if iPTH was >150 pg/mL and serum calcium was <9.8 mg/dL at the dose, which was reduced by 150 mcg per week. For those subjects who experienced an increase in iPTH greater than 100% from pre-treatment baseline, or whose iPTH was greater than 1200 pg/mL at two consecutive visits (at least 2 weeks apart), doses of experimental doses were discontinued. drugs and were excluded from further participation in the treatment period, immediately including them in the follow-up period after treatment.

Key parameters monitored at regular intervals throughout the study include iPTH, serum calcium, serum phosphorus, total serum 25-hydroxyvitamin D, total serum 1,25-dihydroxyvitamin D, serum 25-hydroxyvitamin D3, 1, Serum 25dihydroxyvitamin D3, serum 24,25-dihydroxyvitamin D3, vital signs (VS) and adverse events (AE). Electrocardiograms were recorded at baseline and during the last month of treatment. Additional diagnostic parameters were also monitored, including the content of fibroblast growth factor 23 (FGF-23), the content of bone-specific alkaline phosphatase in serum (BAP), the content of the C-terminal type 1 collagen telopeptide in serum (CTx), the content of the N-terminal propeptide of procollagen type 1 in serum (P1NP), T50 calcification predisposition score, other bone markers and immune function.

Most of the subjects showed a mean decrease in serum iPTH from 30 to 50% relative to baseline before treatment, during the last 6 weeks of treatment (47-52 weeks). Nearly steady state was reached after 12 weeks of treatment (median time to steady state was approximately 16 weeks), with subjects taking the extended release 25-hydroxyvitamin D3 dosage form demonstrating serum 25-hydroxyvitamin D3 concentrations of about 50 to about 90 ng/ml for the 300 µg/week group, about 120 to about 160 ng/ml for the 600 µg/week group, and about 150 to about 200 ng/ml for the 900 µg/week group.

Example 4 Safe Elevation of Serum 1,25-Dihydroxy Vitamin D to Superphysiological Levels.

In FIG. 8 and 9 show preliminary results obtained for two subjects in a trial involving the administration of sustained release oral calcifediol to subjects over a period of up to one year.

The following protocol was used. Subjects took sustained-release oral calcifediol capsules at an initial daily dose of 30 microns, for

-

Claims (14)

four weeks before bed. After the initial dose, the daily dose was increased in increments of 30 μg at intervals of four weeks, up to a maximum dose of 300 μg per day. During dose escalation, if serum calcium was greater than 10.3 mg/dL for two consecutive visits, then dosing was suspended until serum calcium was 10.0 mg/dL or less, at which time dosing to the patient was resumed for 30 mcg less than the previous dose, and maintained the indicated dose level for twelve weeks. Doses were discontinued if serum calcium exceeded 10.3 mg/dl for the third time. Subjects enrolled in the study had a diagnosis of advanced breast or prostate carcinoma with bone stasis and were on current treatment with denosumab or zoledronic acid. The subjects' baseline serum calcium levels were less than 9.8 mg/dL and plasma iPTH levels were greater than 70 pg/mL. In FIG. 8 and 9 show that the serum levels of calcifediol in these subjects increased to about 300 ng/ml or more, and the serum calcitriol levels increased to more than 250 pg/ml. Serum calcium and serum phosphate plots were relatively flat. The ratio of 25-hydroxyvitamin D3 to 24,25-dihydroxyvitamin D3 in the serum of these patients was less than 20, and was typically about 10:1 or less. The foregoing description is provided for clarity of understanding only and is not intended to be undue restrictive, as modifications within the scope of the present invention may be understood by those skilled in the art. In the text of the present description and the following claims, unless the context otherwise requires, the expression contain and its variants, such as contains and containing, should be understood as implying the inclusion of the specified element or step, or group of elements or steps, but not the exclusion of any other element or stages, or groups of elements or stages. In the text of the present description, if compositions are described as containing components or materials, it is provided that these compositions may also consist essentially of or consist of any combination of the listed components or materials, unless otherwise indicated. Likewise, if the methods are described as including certain steps, it is contemplated that said methods may also consist essentially of or consist of any combination of the steps listed, unless otherwise indicated. The invention illustratively described herein may be practiced in the absence of any element or step not specifically described herein. The practical implementation of the method described in this document, and its individual stages can be carried out manually and/or using electronic equipment. Although the methods are described with reference to specific implementation options, those skilled in the art will appreciate that other embodiments of the actions associated with these methods can be used. For example, the order in which the various steps are performed can be changed without departing from the scope or spirit of said method, unless otherwise indicated. In addition, some of the individual steps may be combined, omitted, or further subdivided into further steps. All patents, publications and references cited in this document are incorporated herein by reference in their entirety. In the event of any conflict between the present disclosure and the incorporated patents, publications, and references, the present disclosure shall govern. CLAIM 1. A method of treating secondary hyperparathyroidism in a patient with stage 3 or 4 chronic kidney disease, wherein the ratio of serum 25-hydroxyvitamin D to serum 24,25-dihydroxyvitamin D is greater than 20 and the serum 25-hydroxyvitamin D level is at least 30 ng / ml, which includes the administration of repeated doses of 25-hydroxyvitamin D to the specified patient, effective to increase the concentration of 25-hydroxyvitamin D in the patient's serum to a value of more than 90 ng / ml and to regulate the ratio of 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D in patient serum to a value less than 20. 2. The method of claim 1 wherein repeated doses are administered over an extended period of time of at least one month. 3. The method according to claim 1 or 2, wherein repeated doses are administered for at least 3 months. 4. The method of any one of claims 1 to 3, wherein repeated doses are effective in increasing the serum concentration of 25-hydroxyvitamin D in the patient to a value greater than 100 ng/mL. 5. The method of any of claims 1 to 4, wherein said amount is effective to control the ratio of 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D in the patient's serum to less than 15. 6. The method of any of claims 1 to 5, wherein said doses of 25-hydroxyvitamin D are administered as controlled release oral dosage forms. 7. The method according to any of the preceding paragraphs, including the introduction of a dosage form for - 21 041576 oral administration of sustained release calcifediol. 8. The method of any one of the preceding claims, comprising administering a sustained release dosage form of 25hydroxyvitamin D containing about 30 μg of 25hydroxyvitamin D ₃ , a controlled release waxy agent, an emulsifier, an absorption enhancer, an oleaginous carrier, and a stabilizing agent, said dosage form upon administration a patient with stage 3 or 4 CKD after repeated dosing has one or more of the following pharmacokinetic parameters adjusted for baseline: (A) 25hydroxyvitamin D ₃ after repeated dosing: (1) Cmax of about 10 to about 45 ng/mL and (2) AUC ₀ _ _{6 weeks} from about 300 to about 1300 ng-d/ml; and optionally further Tmax from about 8 to about 45 days, or (B) 1,25-dihydroxyvitamin D after repeated doses: (1) Cmax from about 1 to about 21 pg/ml and (2) AUC ₀ _ _{6 weeks} from about 1 to about 460 g-d/ml, additionally optional Tmax from about 1 to about 44 days. 9. The method of any one of the preceding claims, comprising administering a sustained release dosage form of 25hydroxyvitamin D containing about 60 μg of 25hydroxyvitamin D3, a controlled release waxy agent, an emulsifier, an absorption enhancer, an oleaginous carrier, and a stabilizing agent, said dosage form, upon administration a patient with stage 3 or 4 CKD after repeated dosing has one or more of the following pharmacokinetic parameters adjusted for baseline: (A) 25hydroxyvitamin D ₃ after repeated dosing: (1) Cmax of about 30 to about 90 ng/mL and (2) AUC ₀ _ _{6 weeks} from about 700 to about 2300 ng-d/ml; and optionally further Tmax from about 29 to about 45 days, or (B) 1,25-dihydroxyvitamin D after repeated doses: (1) Cmax from about 7 to about 30 pg/ml and (2) AUC ₀ _ _{6 weeks} from about 1 to about 570 g-d/ml, additionally optional Tmax from about 8 to about 44 days. 10. A method according to any one of the preceding claims, comprising administering a sustained release dosage form of 25hydroxyvitamin D containing from about 10 to about 90 micrograms of 25-hydroxyvitamin D ₃ , a controlled release waxy agent, an emulsifier, an absorption enhancer, an oily carrier, and a stabilizing agent, patient with stage 3 or 4 CKD, comprising administering to the patient repeated doses of an extended release oral dosage form effective to achieve one or more of the following pharmacokinetic parameters, adjusted for baseline: (A) 25-hydroxyvitamin D3: (1) Cmax from about 5 to about 150 ng/ml; (2) Tmax from about 5 to about 60 days; (3) AUC ₀ _ _{6 weeks} from about 100 to about 3300 ng-d/ml, or (B) 1,25-dihydroxyvitamin D: (1) Cmax from about 0.1 to about 50 pg/ml; (2) Tmax from about 1 to about 44 days; (3) AUC ₀ _ _{6 weeks} from about 1 to about 1300 g-d/ml. 11. The method of any one of the preceding claims, comprising administering a sustained release dosage form of 25hydroxyvitamin D containing about 30 μg of 25hydroxyvitamin D ₃ , optionally containing a controlled release waxy agent, an emulsifier, an absorption enhancer, an oily carrier, and a stabilizing agent, to a patient in need of this comprising administering to said subject repeated doses of an extended release oral dosage form effective to achieve one or more of the following: (1) an increase in mean serum 25-hydroxyvitamin D of about 1 to about 7 ng/ml a week; (2) an increase in mean serum 1,25-dihydroxyvitamin D of about 1 to about 10 pg/mL/week. 12. The method of any one of the preceding claims, comprising administering a sustained release dosage form of 25-hydroxyvitamin D containing about 30 to about 60 micrograms of 25-hydroxyvitamin D3, optionally containing a controlled release waxy agent, an emulsifier, an absorption enhancer, an oily carrier, and a stabilizing agent , to a patient in need thereof, comprising administering to said subject repeated doses of a sustained release oral dosage form effective to achieve one or more of the following: (1) a reduction in intact plasma PTH from baseline of about 30 to 80 pg/ml; (2) an increase in serum 1,25-dihydroxyvitamin D from baseline of at least about 10 pg/mL; (4) an increase in serum calcium from baseline of 0 to about 0.3 mg/dl; (5) an increase in serum phosphorus from baseline of 0 to about 0.3 mg/dl; (6) a decrease in serum BSAP from baseline of at least about 10 U/L; (7) a decrease in serum CTX-1 from baseline of at least about 100 pg/mL; and (8) a decrease in serum P1NP from baseline of at least about 30 ng/mL. 13. The method of any one of the preceding claims, wherein repeated doses are effective in lowering the patient's plasma PTH concentration by at least 15%, optionally by at least 30%, relative to baseline. 14. The method according to any of the preceding paragraphs, in which repeated doses are administered periodically -