**8. Treatment of HPP based on the pathophysiology of the disease**

There have been several trials for the treatment of HPP. Respiratory aid somehow succeeds in saving life in the perinatal and the infantile forms, although it is a symptomatic treatment. Other symptomatic treatments are diet therapies, including calcium restriction and vitamin D supplementation, and surgical operations for bone fractures and craniosynostosis [1]. In terms of treatment based on the pathophysiology of HPP, ERT has been attempted. Whyte et al. used the serum of Paget's disease patients who exhibited a high level of TNAP for enzyme replacement [64]. Infusion of PLAP has also been attempted based on the observation that, when the patients with mild forms become pregnant, which causes a high serum ALP level according to an increase in PLAP, they sometimes show improvement of symptoms. Those ERT attempts, however, failed to improve the symptoms [3]. Bone marrow transplantation (BMT) and mesenchymal cell transplantation have also been attempted. Those trials showed a slight improvement but an insufficient effect [65]. Successful ERT was reported in 2012, in which bioengineered TNAP was administered [7]. The C-terminal membrane-bound region of human TNAP was eliminated and replaced with the Fc region of human IgG and deca-aspartate sequences [66]. This bioengineered TNAP is, therefore, soluble, can be easily purified using the Fc region, and has high affinity for hydroxyapatite through acidic peptides such as deca-aspartate [66]. Before the trial, an animal experiment using the bioengineered TNAP in a knockout mouse (*Akp2*−/−; *AKP2* is the mouse homolog of the *ALPL*) that is a good mimic of the perinatal form of HPP, showed elongation of life and improvements in bone and dental defects without respiratory failure [66, 67]. The clinical trial with the bioengineered TNAP (ENB-0040; asfotase alfa) was conducted with five perinatal and six infantile patients [7]. It was administered first as a single intravenous infusion of 2 mg/kg, which was then followed by subcutaneous injections three times per week at a dose of 1 mg/kg for 48 weeks. With the exception of one case who died of respiratory failure that was unrelated with asfotase alfa, the recruited patients showed improvements in rickets and respiratory failure [7]. Asfotase alfa (StrensiqST; Alexion Pharmaceuticals, Inc.) was approved in Japan, the EU, Canada, and the USA in that order in 2015 [2]. Asfotase alfa has dramatically changed the treatment of HPP [68]. Asfotase alfa is indicated for the treatment of patients with perinatal-, infantile- and juvenile-onset HPP [69], in which juvenile-onset HPP means almost the same as the childhood form. The current protocol of the recommended administration is subcutaneous injection six times a week at a dose of 1 mg/kg or three times a week at 2 mg/kg, and the maximal volume of injection is 1 ml [69]. The half-life of asfotase alfa is 5 days in the case of subcutaneous administration. The most common adverse reactions (≥ 10%) are injection site reactions, lipodystrophy, ectopic calcifications, and hypersensitivity reactions. Patients with HPP are at increased risk for developing ectopic calcifications, especially of the eye including the cornea and conjunctiva, and the kidneys (nephrocalcinosis). Although ectopic calcification of the blood vessels has not been reported, it is conceivable that long -term administration may cause medial artery calcification. Medial artery calcification or Mönckeberg-type calcification is often shown as a lethal complication in chronic kidney disease (CKD) patients [70]. In CKD patients, hyperphosphatemia triggers transformation of smooth muscle cells in the media into osteoblastic cells that express elevated TNAP, which then stimulates calcification in the medial artery by a mechanism similar to that of bone mineralization [71, 72]. Asfotase alfa is still not indicated for milder form HPP patients. In this regards, the natural history of the adult form has not been well elucidated [44], and more study is needed. Similarly, odontohypophosphatasia may be still underdiagnosed, because dentists usually do not evaluate the serum ALP value. There should be more investigation into the feasibility of using asfotase alfa for those milder forms.

#### **9. Future perspective**

from the combination of the genotypes. Mutation sites are scattered throughout the gene, but there are some "hot spots." In Caucasian patients, p.E191K (a moderate allele with a dominant negative effect) and p.D378V (a severe allele) are frequent mutations [60, 61], whereas c.1559delT (pL520RfsX86; a severe allele) and p.F327L (a moderate allele) are frequent in Japanese patients [62, 63]. c.1559delT also has founder effects, and the frequency of c.1559delT

Mutation sites of TNAP proteins are classified by its domain structure [30]. Severe phenotypes are associated with the mutations that are located in the active site and its vicinity, the homodimer interface, the crown domain, and the calcium-binding domain. Mutations in the active site valley (the entry site of the substrate into the active site) resulted in less severe phenotypes [30]. Mutations in the other regions of the protein are inclined to show residual

Because most of the patients are compound heterozygotes, the residual activity and phenotype are determined by the interaction of two mutant proteins [55]. In some cases, especially in autosomal dominant cases, dominant negative mechanisms are suggested, in which cases the mutant proteins affect the function of the wild-type enzymes [48]. Those interactions have not been precisely elucidated and need to be explored in more detail in order to reveal the

There have been several trials for the treatment of HPP. Respiratory aid somehow succeeds in saving life in the perinatal and the infantile forms, although it is a symptomatic treatment. Other symptomatic treatments are diet therapies, including calcium restriction and vitamin D supplementation, and surgical operations for bone fractures and craniosynostosis [1]. In terms of treatment based on the pathophysiology of HPP, ERT has been attempted. Whyte et al. used the serum of Paget's disease patients who exhibited a high level of TNAP for enzyme replacement [64]. Infusion of PLAP has also been attempted based on the observation that, when the patients with mild forms become pregnant, which causes a high serum ALP level according to an increase in PLAP, they sometimes show improvement of symptoms. Those ERT attempts, however, failed to improve the symptoms [3]. Bone marrow transplantation (BMT) and mesenchymal cell transplantation have also been attempted. Those trials showed a slight improvement but an insufficient effect [65]. Successful ERT was reported in 2012, in which bioengineered TNAP was administered [7]. The C-terminal membrane-bound region of human TNAP was eliminated and replaced with the Fc region of human IgG and deca-aspartate sequences [66]. This bioengineered TNAP is, therefore, soluble, can be easily purified using the Fc region, and has high affinity for hydroxyapatite through acidic peptides such as deca-aspartate [66]. Before the trial, an animal experiment using the bioengineered

is mentioned above [46, 62].

110 Pathophysiology - Altered Physiological States

**7. Structure and function of mutant TNAP**

enzymatic activity and are, therefore, milder phenotypes.

genotype–phenotype interrelationships and pathophysiology of HPP.

**8. Treatment of HPP based on the pathophysiology of the disease**

Although current ERT has drastically changed the treatment of HPP, many problems are indicated regarding asfotase alfa administration. First of all, two or three injections per week are needed for this ERT treatment, which burdens patients with injections and parents with administration fees. The interval between injections can be elongated by introducing some modifications into the enzyme preparation. Other possible therapies are bone marrow stemcell transplantation and/or combination therapy of such transplantation with ERT. Another possible trial is a trial of gene therapy. Using viral vectors, gene therapy was successfully used to treat knockout mice (*AKP2*−/−) [73, 74]. Since a viral vector containing *ALPL* cDNA that is injected into blood cannot maintain an effective concentration, gene therapy in combination with stem-cell transplantation (*ex vivo* gene therapy) may be more effective [75]. Once gene-transferred stem cells are transplanted, no other injection may be necessary [2]. Although gene therapy seems to be a promising procedure, results have so far only been obtained for mouse models, and its feasibility and safety in humans must be investigated.

[2] Orimo H. Pathophysiology of hypophosphatasia and the potential role of asfotase alpha.

Hypophosphatasia: A Systemic Skeletal Disorder Caused by Alkaline Phosphatase Deficiency

http://dx.doi.org/10.5772/intechopen.70597

113

[3] Whyte MP. Hypophosphatasia: An overview for 2017. Bone. 2017;**102**:15-25. DOI:

[4] Rathbun JC. "Hypophosphatasia": A new developmental anomaly. American Journal of

[5] Mumm S, Jones J, Finnegan P, Whyte MP. Hypophosphatasia: Molecular diagnosis of Rathbun's original case. Journal of Bone and Mineral Research. 2001;**16**:1724-1727

[6] The Tissue-Nonspecific Alkaline Phosphatase Gene Mutations Database [Internet]. Available from: http://www.sesep.uvsq.fr/03\_hypo\_mutations.php [Accessed: 31 May

[7] Whyte MP, Greenberg CR, Salman NJ, Bober MB, McAlister WH, Wenkert D, Van Sickle BJ, Simmons JH, Edgar TS, Bauer ML, Hamdan MA, Bishop N, Lutz RE, McGinn M, Craig S, Moore JN, Taylor JW, Cleveland RH, Cranley WR, Lim R, Thacher TD, Mayhew JE, Downs M, Millán JL, Skinar AM, Crine P, Landy H. Enzyme-replacement therapy in life-threatening hypophosphatasia. New England Journal of Medicine. 2012;**366**:904-913

[8] Orimo H. The mechanism of mineralization and the role of alkaline phosphatase in

[9] Millán JL. Mammalian Alkaline Phosphatases: From Biology to Applications in Medicine and Biotechnology. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co.; 2006

[10] Harris H. The human alkaline phosphatases: What we know and what we don't know.

[11] Smith M, Weiss MJ, Griffin CA, Murray JC, Buetow KH, Emanuel BS, Henthorn PS, Harris H. Regional assignment of the gene for human liver/bone/kidney alkaline phos-

[12] Weiss MJ, Ray K, Henthorn PS, Lamb B, Kadesch T, Harris H. Structure of the human liver/bone/kidney alkaline phosphatase gene. Journal of Biological Chemistry. 1988;

[13] Matsuura S, Kishi F, Kajii T. Characterization of a 5′-flanking region of the human liver/bone/kidney alkaline phosphatase gene: Two kinds of mRNA from a single gene.

[14] Kiledjian M, Kadesch T. Analysis of the human liver/bone/kidney alkaline phosphatase

[15] Orimo H, Shimada T. Regulation of the human tissue-nonspecific alkaline phosphatase gene expression by all-*trans*-retinoic acid in SaOS-2 osteosarcoma cell line. Bone.

Biochemical and Biophysical Research Communications. 1990;**168**:993-1000

promoter *in vivo* and *in vitro*. Nucleic Acids Research. 1990;**18**:957-961

health and disease. Journal of Nippon Medical School. 2010;**77**:4-12

phatase to chromosome 1p36.1-34. Genomics. 1988;**2**:139-143

Therapeutics and Clinical Risk Management. 2016;**12**:777-786

10.1016/j.bone.2017.02.011

2017]

Diseases of Children. 1948;**75**:822-831

Clinica Chimica Acta. 1989;**186**:133-150

**263**:12002-12010

2005;**36**:866-876
