Sklerostin

Human sclerostin (Scl) - protein of SEQ ID NO:1

• Product of the SOST gene
• Absent in sclerosteosis
• Skeletal disease
• Bone overgrowth and strong dense bones [1]

Funkce sklerostinu

• Potent inhibitor of osteoblastogenesis [4]
• Glycoprotein secreted by osteocytes
• Travels through osteocyte canaliculi to the bone surface
• Binds to coreceptors LRP5 and LRP6
• preventing colocalization with frizzled protein and Wnt signaling
• Reducing osteoblastogenesis and bone formation [4]
• Inhibice mineralizace osteoblastů [1]
• Glucocorticoids inhibit developmental stage-specfic osteoblast cell cycle [1]
• Sclerostin inhibits mineralization [1]

Mutace SOST genu pro sklerostin

• Ztráta funkce
• autosomal-recessive disorder - sclerosteosis
• Progressive bone overgrowth [4]
• Generalized osteosclerosis with skeletal deformities [5]
• Cranial nerve compression [5]
• Increased intracranial pressure due to boney overgrowth in the skull [5]
• Premature death [5]
• Heterozygoté
• Normal phenotype and normal lifespan [5]
• Dense bones and low risk of fracture [5]
• Reduced sclerostin expression
• Deletion downstream of the SOST gene -
• Milder form of van Buchem disease [4]
• SOST-null mice
• High-bone-mass phenotype [4]
• SOST-knockout mice were resistant to mechanical unloading bone loss [4]

Hladiny sklerostinu

• Sclerostin suppression
• Balanced remodeling in response to PTH [4]
• Sclerostin levels significantly higher
• In postmenopausal women
• Significant negative correlations between free estrogen levels and sclerostin as well as PTh and sclerostin [4]
• Sclerostin gene expression and protein levels reduced
• In animals treated with daily injections of human parathyroid hormone (hPTH) [4]

Monitorace kostní novotvorby

Various serum anabolic markers

• Osteocalcin
• P1NP (n-terminal propeptide of type 1 procollagen)

Histomorphometric markers of bone formation

• Osteoblast surface/bone surface
• Bone formation rate/bone surface
• Trabecular thickness
• Bone mineral density
• Bone mineral content
• Bone mass
• Bone quality
• Bone strength [1]

Increases in bone mineral content/ density

• X-rays
• Dual Energy X-ray Absorptometry
• "DEXA" [1]

Serum markers of bone formation / osteoblast activity

• Osteoblast specific alkaline phosphatase
• Osteocalcin
• Type 1 procollagen C' propeptide (PICP)
• Total alkaline phosphatase
• Serum TRAP 5b (tartrate-resistant acid phosphatase isoform 5b)
• Serum cross-linked C-telopeptide (sCTXI) [1]

Amount of bone mass

• Calculated from body weights
• Others [1]

Riziko osteoporozy

• The lifetime risk of an osteoporotic fracture is about 50% for Caucasian women and 20% for men (U.S. Department of Health and Human Services, 2004) [5]
• Každá 2. žena v USA !!!!!!!!
• Fractures of the spine and hip are associated with acute and chronic pain, deformity, depression, disability, and increased risk of death (Kanis and on behalf of the World Health Organization Scientific Group, 2007) [5]

Sclerostin binding antibodies - AMG 785 (CDP7851) sub-cutaneous injections

• Bind to the polypeptide fragments of sclerostin (neutralisation)

Antibodies are useful in:

• Diseases of low bone density - osteoporosis [1]
• Accelerating the healing of bone fractures [1]
• To block or impair binding of human sclerostin to one or more ligand [1]
• Neutralize sclerostin's inhibitory activity
• Allow for mineralization of the culture in the presence of sclerostin
• Statistically significant increase in deposition of calcium phosphate (measured as calcium) compared to the amount of calcium measured in the sclerostin-only (i.e. no antibody) [1]

Možné indikace

• Dysplasias
• Growth or development of bone is abnormal
• Wide variety of causes of osteopenia, osteoporosis and bone loss
• Achondroplasia
• Cleidocranial dysostosis
• Enchondromatosis
• Fibrous dysplasia
• Gaucher's Disease
• Hypophosphatemic rickets
• Marfan's syndrome
• Multiple hereditary exotoses
• Neurofibromatosis
• Osteogenesis imperfecta
• Osteopetrosis
• Osteopoikilosis
• Sclerotic lesions
• Pseudoarthrosis
• Pyogenic osteomyelitis
• Periodontal disease
• Anti-epileptic drug induced bone loss
• Primary and secondary hyperparathyroidism
• Familial hyperparathyroidism syndromes
• Weightlessness induced bone loss
• Osteoporosis in men
• Postmenopausal bone loss
• Osteoarthritis
• Renal osteodystrophy
• Infiltrative disorders of bone
• Oral bone loss
• Osteonecrosis of the jaw
• Juvenile Paget's disease
• Melorheostosis
• Metabolic bone diseases
• Mastocytosis
• Sickle cell anemia/disease
• Organ transplant related bone loss
• Kidney transplant related bone loss
• Systemic lupus erythematosus
• Ankylosing spondylitis
• Epilepsy
• Juvenile arthritides
• Thalassemia
• Mucopolysaccharidoses
• Fabry disease
• Turner syndrome
• Down Syndrome
• Klinefelter Syndrome
• Leprosy
• Perthes' Disease
• Adolescent idiopathic scoliosis
• Infantile onset multi-system inflammatory disease
• Winchester Syndrome
• Menkes Disease
• Wilson's Disease
• Ischemic bone disease
• Legg-Calve-Perthes disease
• Regional migratory osteoporosis
• Anemic states
• Conditions caused by steroids
• Glucocorticoid induced bone loss
• Heparin - induced bone loss
• Bone marrow disorders
• Scurvy
• Malnutrition
• calcium deficiency
• Idiopathic osteopenia or osteoporosis
• Congenital osteopenia or osteoporosis
• Alcoholism
• Chronic liver disease
• Postmenopausal state
• Chronic inflammatory conditions
• Rheumatoid arthritis
• Inflammatory bowel disease
• Ulcerative colitis
• Inflammatory colitis
• Crohn's disease
• Oligomenorrhea
• Amenorrhea
• Pregnancy
• Diabetes mellitus
• Hyperthyroidism
• Thyroid disorders
• Parathyroid disorders
• Cushing's disease
• Acromegaly
• Hypogonadism
• Immobilization or disuse
• Reflex sympathetic dystrophy syndrome
• Regional osteoporosis
• Osteomalacia
• Bone loss associated with joint replacement
• HIV associated bone loss
• Bone loss associated with loss of growth hormone
• Bone loss associated with cystic fibrosis
• Fibrous dysplasia
• Chemotherapy associated bone loss
• Tumor induced bone loss
• Cancer - related bone loss
• Hormone ablative bone loss
• Multiple myeloma
• Drug - induced bone loss
• Anorexia nervosa
• Disease associated facial bone loss
• Disease associated cranial bone loss
• Disease associated bone loss of the jaw
• Disease associated bone loss of the skull
• And bone loss associated with space travel
• Associated with aging
• Facial bone loss associated with aging
• Cranial bone loss associated with aging
• Jaw bone loss associated with aging
• And skull bone loss associated with aging [1]
• Useful for improving outcomes in
• Orthopedic procedures
• Dental procedures
• Implant surgery
• Joint replacement
• Bone grafting,
• Bone cosmetic surgery
• Bone repair such as
• Fracture healing
• Nonunion healing
• Delayed union healing
• Facial reconstruction

Výroba

• Rekombinantní cestou tvorba fragmentů lidských protilátek schopných vazby na slerostin
• Escherichia coli
• Saccharomyces cerevisiae
• Schizosaccharomyces pombe
• Pichia pastoris
• Mammalian cells
• Myeloma (such as a mouse NSO line), COS, CHO, or hybridoma cells
• Plant cells
• Tobacco, corn, soybean, and rice cells [1]
• Conventional immunization
• By injecting an animal, for example, a rat, hamster, a rabbit, or preferably a mouse [1]
• Cell fusion procedures
• Lymphoid cells, most commonly cells from the spleen or lymph node, are removed to obtain B-lymphocytes [1]
• Lidské monoklonální protilátky
• Epstein Barr Virus (EBV) transformation of human peripheral blood cells
• B lymphocytes
• In vitro immunization of human B cells
• Fusion of spleen cells from immunized transgenic mice carrying inserted human immunoglobulin genes
• Isolation from human immunoglobulin V region phage libraries [1]
• Other [1]

Možné pomocné látky injekčních forem

Možné konzervanty

• Parabens
• Chlorobutanol
• Phenol
• Sorbic acid
• Thimerosal
• A pod. [1]

Isotonic agents

• sugars
• Sodium chloride apod. [1]

Prolonged absorption

• Aluminum monostearate
• Gelatin
• A pod. [1]

Míra neutralizace protilátek

• Very potent anti-sclerostin antibody
• Neutralize sclerostin even when there is less than a 6-fold excess of moles of sclerostin binding sites per well [1]
• Less potent anti-sclerostin antibody
• Neutralize sclerostin only at a 12,18 or 24 fold excess of binding sites [1]

Pokusy na zvířatech

Myši

• Increase in bone mass at all skeletal envelopes, including cancellous, cortical bone sites, and supervertebral sites
• Increase in bone mass
• Change in microarchitecture
• Improved bone strength in both the appendicular and axial skeleton
• Sclerostin antibody + zoledronic acid resulted in additive effects on bone parameters and bone mass [4]
• Not gender specific [4]

• Mouse model of colitis, short-term treatment
• Increased bone strength and bone formation
• Rodent models of fracture healing
• Increased callus density
• Incr. bone strength at fracture sites
• Accelerated bone repair [4]

• Scl-AbI is an anti-sclerostin monoclonal antibody that has been shown to stimulate Wnt/ß-catenin signaling in cell culture and increase BMD in mice (Veverka et al., 2009) [5]

Cynomolgus monkeys

• Gonad-intact female
• Dose-dependent increases in bone formation
• Trabecular
• Periosteal
• Endocortical
• Intracortical [4]
• Bone density
• Significant increases
• Femoral neck
• Radial and tibial metaphysis
• Trabecular thickness and bone strength in the lumbar vertebrae [4]
• Improved strength of the fracture callus in a primate fibular osteotomy model [4]

Klinické studie na téma sklerostin registrované v USA

A First-in-human Study Evaluating AMG 785 in Healthy Men and Postmenopausal Women

• Phase 1 Clinical Trial
• In 72 healthy men and postmenopausal women [5]
• A Randomized, Double-blind, Placebo-controlled
• Ascending Single-dose Study to Evaluate the Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of AMG 785 in Healthy Men and Postmenopausal Women
• Postmenopausal women received AMG 785 or placebo
• Weight-based dose of
• 0.1 mg/kg, 0.3, 1, 3, 5, or 10 mg/kg SC or
• 1 mg/kg or 5 mg/kg IV [2]
• Men will received doses
• 1 mg/kg or 5 mg/kg SC or IV [2]
• Start Date - December 2006
• Verification Date May 2010 [2]
• Padhi et al., 2011 [5]

A Single-dose Study Evaluating AMG 785 in Healthy Postmenopausal Japanese Women

• Verification Date March 2011

Study To Assess Fracture Healing With Sclerostin Antibody

An Ascending Multiple Dose Study Evaluating AMG 167 in Healthy Men and Postmenopausal Women With Low Bone Mineral Density

• Skup. 1 - 6 women
• Skup. 4 - 6 žen + option to receive an additional 6 D
• Skup. 2 - 6 mužů
• AMG 167 á 2 weeks - 6 D

• Skup. 3 - 6 žen
• Skup. 5 - 6 žen + option to receive an additional 3 D
• Skup. 6 - 6 mužů + option to receive an additional 3 D
• AMG 167 á 4 weeks - 3 D

• Skup. 7 - 18 women + option to transition to 6 months of alendronate
• AMG 167 á 2 weeks - 12 D

• Primary Completion Date
• February 2012

Phase 2 Clinical Trial of AMG 785 in Postmenopausal Women with Low BMD

• 419 postmenopausal women with low BMD

Study to Determine the Efficacy and Safety of AMG 785 in the Treatment of Postmenopausal Women With Osteoporosis

• Fáze 3 klinického testování
• Randomized, Safety/Efficacy Study, Parallel Assignment, Double Blind (Subject, Caregiver, Investigator, Outcomes Assessor), Treatment
• Estimated Primary Completion Date
• May 2015 (final data collection date for primary outcome measure)
• Skupiny studie:
• Experimental group: AMG 785
• AMG 785 sub-cutaneous injections + placebo alendronate (oral) for 12 months
• Followed by open-label alendronate (oral) for at least another 12 months (until end of study)
• Active Comparator group: Alendronate
• Oral alendronate + placebo AMG 785 sub-cutaneous injections for 12 months,
• Followed by open-label alendronate (oral) for at least another 12 months (until end of study) [3]
• Study Sponsor: Amgen [3]
• Studie probíhá také v ČR

Farmakokinetika atd.

• Renal elimination is insignificant for monoclonal antibodies, since the molecules are too large for glomerular filtration. [5]

Potencionální možné nežádoucí účinky terapie k evaluaci

• Vznik carpal tunnel syndrome [4]
• Lumbar radiculopathy [4]
• Spinal stenosis [4]
• SOST mRNA expression in off-target tissues, such as kidney, heart, and liver (Brunkow et al., 2001) [5]
• Suggests the possibility of unintended non-skeletal effects with anti-sclerostin therapy [5]
• Sclerostin protein has not been detected postnatally in any of these organs [5]

NÚ v 1. fázi klinických studií (72 zdravých jedinců)

• Generally well tolerated
• 1 z NÚ u 25% - 60% dle dávky
• Nejč. NÚ - mild and not serious
• Site erythema
• Back pain
• Headache
• Constipation
• Injection site hemorrhage
• Arthralgia
• Dizziness [5]
• One serious NÚ - hepatitida
• člověk received 10 mg/kg SC AMG 785
• Severe non-specific hepatitis [5]
• Elevated liver function test beginning 1 day after dosing
• Liver enzymes peaking at levels of 6-13 times the upper limit of normal
• Six to 8 days after dosing, abdominal ultrasound tests and hepatitis panels were normal
• No additional information was provided [5]
• Mild, transient asymptomatic NÚ
• Decreases in mean serum ionized calcium levels (about 4% below baseline) returning to baseline [5]
• Transient increase in serum intact PTH levels that returned to baseline
• Higher doses of AMG 785 vedly k increased total serum alkaline phosphatase
• Finding of neutralizing antibodies [5]
• Not associated with any NÚ or abnormalities of other laboratory tests, vital signs, or electrocardiogram [5]

NÚ v Phase 2 Clinical Trial

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