A farewell to phlebotomy—use of placenta-derived drugs Laennec and Porcine for improving hereditary hemochromatosis without phlebotomy: a case report

Iron concentration in the body is rigorously regulated. The intracellular supply and storage of iron is mediated principally by three proteins: transferrin (Tf), TfR, and FT. Tf is a serum glycoprotein responsible for transporting bound iron from the absorbed organs to the peripheral tissues [31]. Furthermore, Tf, a component of an iron-sensing system, is essential for the maintenance of iron homeostasis by modulating hepcidin production [32]. Most cells modulate iron uptake by regulating the expression and dynamics of TfR. Regulation is mediated by intracellular iron levels and iron-responsive elements within the cell: mRNAs that are recognized by special iron regulatory proteins [31]. Hepcidin, a principal regulator of iron, is secreted into the bloodstream and interacts with enterocytes to adjust the rate of iron absorption [9, 33]. Bone morphogenetic protein (BMP)–SMAD (SMA and mothers against decapentaplegic homolog) signaling is modulated by various cofactors, including positive factors, such as TfR2, HFE, and Hjv, and negative factors, such as TMPRSS6 and soluble Hjv. The expression of BMP6 mRNA was recently shown to increase with iron loading in mice, suggesting that BMP6 is a signal that reflects the iron stores [34]. BMP–SMAD signaling is known to regulate hepcidin synthesis. BMPs are cytokines belonging to the TGF superfamily that exist primarily and abundantly in placental tissues. They play crucial roles in regulating cell proliferation, differentiation, and apoptosis throughout the development of each tissue [35]. Recent studies suggest that hepcidin, a peptide involved in iron homeostasis, is regulated by BMPs, apparently by binding to Hjv as a coreceptor and signaling through the SMAD4. The TfR2/HFE complex is also required for transcriptional regulation of hepcidin by the holo-Tf.

With the use of placenta-derived drugs as substitutes to phlebotomy, and based on the evidence from HAMP assays using HepG2 cells and primary hepatocyte cultures shown in Fig. 1a, b, one of the possible mechanisms and surmised sites of action inducing the expression of hepcidin by placenta-derived extracts might be the stimulation and/or acceleration of the BMP–SMAD–Hjv axis, which is located downstream of the intrahepatic iron-sensing system.

Hepatic fibrosis is a complicated dynamic process caused by hepatocyte death and activation of hepatic stellate cells (HSCs). Lipid peroxidation, including the generation of ROS, TGF-β, and TNF-α, is a cause of hepatic fibrosis. HSCs are regarded as the primary target cells that elicit an inflammatory response and produce extracellular matrix components. HSCs are activated by several factors, such as ROS, lipid peroxidation products [malonaldehyde and 4-hydroxynonenal], and TGF-β, which is released when adjacent cells, including hepatocytes, Kupffer cells, and endothelial cells, are injured by various types of disadvantageous pathognomonic abnormalities. The inflammatory generation of Reactive oxygen species (ROS) depends on the Fenton reaction catalyzed by redox-active metals, of which iron predominates. Iron overload causes oxidative stress and subsequent mitochondrial and DNA damage, lipid peroxidation, and protein modification, leading to the dysfunction of metabolically active inflated cells, such as cardiomyocytes, hepatocytes, pituitary cells, chondrocytes, and pancreatic β-cells [36]. In HFE-related HH, juvenile HH, and type 3 HH, iron is typically stored in periportal hepatocytes, with little or no iron stores in the Kupffer cells. As iron accumulation continues, the midzonal and centrilobular hepatocytes along with the biliary epithelium will progressively accumulate iron [37].

New data from a recent study suggest that impaired BMP signaling underlies hepcidin deficiency in HFE-related HH. Moreover, the inhibitory SMADs, SMAD6 and SMAD7, have been identified as potential disruptors of this signal and, hence, contribute to the pathogenesis of this disease [38]. Although it is classically regarded that phlebotomy cannot reverse liver cirrhosis, diabetes, or hypogonadism [39], our clinical data (Figs. 3, 4, 6, and 8) do not agree with these assumptions. By reducing iron-related ROS, which induce liver fibrosis and cause other organic injuries, the administration of Laennec (parenteral) and Porcine (oral) seems to treat these complications (diabetes and hypogonadism) in HH patients and other related pathological disorders (liver fibrosis and iron deposition in the hepatocytes).

Phlebotomy (venesection therapy) is the standard treatment for patients with HH and has been used for more than 70 years. It is effective in reducing the morbidity and mortality rates among HH patients [28]. Iron-depleted HFE C282Y patients have extremely low serum hepcidin concentrations, reflecting the combined effects of HFE mutation and phlebotomy on hepcidin expression [40]. These patients demonstrated an exaggerated increase in the serum iron concentration upon oral iron loading. This observation indicates that reducing the hepcidin concentrations by phlebotomy in HH patients may exacerbate the underlying cause of iron overload, enhancing the excess release of iron into the circulation, with an ensuing “vicious cycle” that leads to the need for more frequent maintenance phlebotomies in HH patients [40]. Correspondingly, intestinal iron absorption is greatly increased in patients with hemochromatosis, even when the iron stores are depleted by phlebotomy. It is important to continue phlebotomies even if the iron stores are depleted to prevent the reaccumulation of iron. However, phlebotomy cannot reverse liver cirrhosis, diabetes, destructive arthritis, cardiomyopathy, or hypogonadism, which are the major complications of HH [39]. Although phlebotomy is specifically performed to remove excess “iron” in the blood, it can also reduce the amount of multiple nutrients, several kinds of essential hormones, and immune-related elements primarily contained in the blood of HH patients. Due to this inefficient and wasteful procedure, the QOL of HH patients tend to deteriorate, not only during the induction phase but also during the maintenance periods.

Persistent iron overload can generally lead to the development and progression of diabetes by lowering insulin production and promoting insulin resistance [41]. In addition to the apparent iron overload in hepatocytes, an increase in the body iron pool can also cause insulin resistance, metabolic syndrome, and gestational diabetes [42]. This assumption is empirically endorsed and based on the following observations: First, reduction of body iron pool by bloodletting or blood donation improves glycemic control and insulin resistance in patients with T2DM [43]; second, chelation therapy and blood donation lower the risk of diabetes in healthy individuals as well as in those with mildly increased body iron stores; and third, iron deficiency enhances insulin sensitivity and lowers the risk of diabetes [44]. Iron overload causes insulin deficiency by promoting pancreatic β-cell apoptosis. Because of their stringent dependence on mitochondrial glucose metabolism and their limited antioxidant capacity [45], β-cells are extremely susceptible to oxidative stress. Through their divalent metal transporter, pancreatic β-cells avariciously take up non-Tf-bound iron [46], which can promote oxidative stress by catalyzing the Fenton reaction. Diabetes is a one of the classic symptoms of HH, and some studies reported that the prevalence of diabetes in people with HH aged 45 years and older exceeds 20% [47]. Several studies have shown an association between hemochromatosis and T2DM [10, 48]. Elevated iron levels oxidize various biomolecules, such as nucleic acids, proteins, and lipids, which may contribute to the development of T2DM by decreasing insulin secretion from pancreatic β-cells, with concomitant increase in insulin resistance [3, 49]. With regard to the efficacy of bloodletting on T2DM, it is generally regarded that phlebotomy as a method used for achieving iron depletion does not improve diabetes control in all people with HFE-related hemochromatosis [50].

In the first 24 months of Laennec treatment (Fig. 2), the HbA1c level has also improved from 8.8% to 7.5% without increasing the dose of insulin, and has further improved from 8.8% to 6.8% for 50 months without changing the medications for diabetes treatment (Fig. 7). These results are presumably due to the additive efficacy of Laennec in reducing iron-originated ROS, enhancing the antiinflammatory action with concomitant improvement in liver fibrosis, and diminishing the iron deposition in the hepatocytes. Laennec was also administered in nonalcoholic steatohepatitis (NASH) patients with T2DM; treatment with Laennec significantly improved the T2DM, reduced the serum ferritin level, and decreased the iron deposition in the hepatocytes [51, 52]. The regulation of iron and glucose metabolism is possibly due to the pancreatic β-cells’ ability to co-release insulin and hepcidin. Even under pathophysiological conditions, there seemed to be a link between the secretion of insulin and that of hepcidin, since patients with HH not only exhibited a decrease in insulin secretion [53] but also a reduction in serum hepcidin levels [54]. As for the hepcidin expression in the pancreas, data published by Kulaksiz et al. [24] demonstrated that hepcidin is expressed in the pancreas of rats and humans. Further analysis showed that it was localized in the β-cells of the islets of Langerhans. In addition, the in vitro experiments performed in this study demonstrated that hepcidin expression in β-cells is directly regulated by iron. Iron is important for normal insulin secretion. However, excessive amounts of iron can affect the β-cell function in hemochromatosis models [26, 27, 55], causing iron accumulation in the islets, a reduction in insulin secretion, and an increase in the apoptosis of β-cells. By contrast, a decrease in the iron pool was shown to protect against diabetes and loss of β-cell function in an obese (ob/ob) mouse model [56]. These observations suggest that hepcidin produced by β-cells may be involved in the intrinsic regulation of pancreatic iron and glucose homeostasis [11]. Iron overload is a risk factor for diabetes. The link between iron and diabetes was first recognized in patients with HH and thalassemia, but high levels of dietary iron at the same time may enhance the risk of diabetes. Iron plays a direct and causal role in the pathogenesis of diabetes, mediated by both β-cell failure and insulin resistance. Iron also regulates metabolism in most tissues involved in fuel homeostasis, with adipocytes playing an iron-sensing role. The underlying molecular mechanisms mediating these effects are numerous and not completely understood, but include oxidative stress, modulation of adipokines, and intracellular signal transduction systems [33, 57]. Iron deposition also induces insulin resistance by inhibiting glucose uptake in fat and muscle tissues, and by reducing the capacity of the liver to extract insulin, which results in an abnormal increase in hepatic glucose production [58].

It seems reasonable to consider that one possible mechanism for the efficacy of Laennec in improving T2DM was the induction of the action of “local hepcidin” from pancreatic β-cells. However, further examinations are necessary to clarify the pharmacological dynamics of placenta-derived drugs.

Serum ferritin is an independent predictor of histologic severity and advanced fibrosis in patients with nonalcoholic fatty liver disease (NAFLD). NAFLD is now recognized as a major cause of chronic liver diseases, including liver cirrhosis and hepatocellular carcinoma (HCC) in Western countries [47]. With the recent progress in the understanding of iron metabolism in patients with HH at the molecular level, accumulating evidence suggests a link between altered iron metabolism and NAFLD. In the last decade, many studies have found an intimate relationship between hepatic iron and NASH or its progression [3, 59, 60]. When iron accumulates, it promotes oxidative free radical reactions, which have harmful effects on multiple organs. In HH, the accumulation of iron in the liver, heart, and pancreas leads to cirrhosis, heart failure, and diabetes, respectively. Even mild iron overload might aggravate insulin resistance, diabetes, atherosclerosis, colonic neoplasia, and NAFLD [51, 52, 57, 59]. Increased oxidative stress is widely regarded as a key factor in the progression and deterioration of chronic liver diseases [49]. Recently, hepatic iron overload has attracted attention because excessive iron accumulation causes severe cellular dysfunction by facilitating the production of ROS, and the liver, which is the principal organ for iron storage, is readily damaged by iron-induced ROS. Moreover, this phenomenon has been frequently observed in patients with both hepatitis C virus infection and Non-alcoholic steatohepatitis (NASH) [49]. Patients with NAFLD frequently experience elevations in serum TS, SF, or both, and elevated serum iron markers may possibly indicate an iron overload [50]. Previous studies [51, 52, 59, 60] revealed that the administration of Laennec significantly improved T2DM complicated with NASH and other chronic liver diseases, which suggests the importance of iron regulation on insulin-resistant T2DM showing hyperferritinemia [51, 52, 59, 60]. A high level of Serum-Ferritin (SF) is a component of insulin resistance (IR). High ferritin levels in IR patients are mainly the result of a chronic inflammatory state induced by NASH and complicated with a metabolic syndrome [61]. Reciprocally, iron interferes with the action of insulin in the liver [62]. In addition, iron is a potent pro-oxidant that increases cellular oxidative stress, causing inhibition of insulin internalization and action, which results in hyperinsulinemia, IR, and abnormal β-cell function due to iron toxicity [59, 60, 63].

In our clinical experience, treatment with Laennec could improve, not only T2DM but also advanced liver fibrosis, even in patients with relatively progressed NASH [51, 52, 59, 60].

Secondary hemochromatosis is usually caused by multiple blood transfusions in patients with hemolytic anemia, such as thalassemia, sickle cell anemia, and myelodysplasia syndrome (MDS). Iron first accumulates in the RES macrophages and is later transferred to the parenchymal cells. With frequent blood transfusions, iron may accumulate faster in patients with genetic hemochromatosis; iron overload often leads to severe cardiomyopathy and liver cirrhosis, limiting the prognosis of iron overload disorders. β-thalassemia is prevalent in Mediterranean countries, the Middle East, Central Asia, India, southern China, and the Far East, as well as in countries along the north coast of Africa and South America [64]. Therapy consists of iron chelators because erythroid disorders, such as β-thalassemia and MDS, are characterized by ineffective hematopoiesis and anemia, and are frequently accompanied by reduced expression of HAMP and followed by iron overload. The current standard of treatment for these disorders includes transfusion and iron chelation. Phlebotomies cannot be performed because of the underlying anemia [65]. However, iron chelators have serious side effects, and transfusions can aggravate iron overload [66]. The mainstay of therapy for β-thalassemia major is lifelong red cell transfusion to improve anemia and suppress ineffective erythropoiesis [67]. Increased erythropoiesis suppresses hepcidin production, resulting in a greater supply of iron from duodenal absorption and iron storage release, allowing sufficient iron to be available for hemoglobin synthesis. However, the molecular mechanisms mediating hepcidin suppression are not well understood [68]. In iron-loading anemia (β-thalassemia and congenital dyserythropoietic anemia), the suppressive effect of erythropoiesis on hepcidin production [69] and the resulting increase in dietary iron absorption can cause systemic iron overload and iron-mediated damage to the liver and myocardium, even without blood transfusions. However, whether treatment with exogenous hepcidin can correct the cause of iron-loading anemias needs to be further elucidated [1]. Patients with thalassemia intermedia experience iron overload even if they do not receive blood transfusions, and their urinary hepcidin levels are remarkably low despite high plasma Tf saturation and systemic iron overload [69]. With the increasing use of transfusion therapy, iron overload has become a major cause of morbidity and premature mortality. More recently, the effective treatment of iron overload by iron chelation has dramatically improved patient survival [70]. In patients with thalassemia, anemia induced by phlebotomy or hemolysis also suppresses the levels of hepatic hepcidin mRNA. Importantly, the suppressive effect of hemolytic anemia was observed even in iron-overloaded mice, suggesting that the suppression of hepcidin levels by anemia has a stronger effect than the stimulation of hepcidin expression by iron overload. This hierarchy of effects could explain why iron overload commonly develops in individuals with certain hemolytic disorders such as thalassemia. However, these observations do not explain why iron overload occurs more commonly in patients with intramedullary hemolysis than in those with peripheral hemolysis or nonhemolytic anemia [8]. In thalassemia intermedia, hepcidin therapy is expected to have beneficial effects by curbing the hyperabsorption of dietary iron. Hepcidin agonists could be useful even in patients with transfused thalassemia. Although intestinal iron absorption contributes less to the total iron load in these patients, hepcidin therapy may help in the period when endogenous hepcidin levels decrease and intestinal iron uptake increases. Hepcidin diagnostics and future therapeutic agonists may help in the management of patients with β-thalassemia [71]. Although these proof-of-principle studies show promise, no hepcidin therapies are yet available. Hepcidin has several characteristics that limit its potential use as a therapeutic agent. Due to its length and complex 4-disulfide structure, it is very expensive to produce.

Probably Laennec and Porcine, which can effectively induce the production of hepcidin, will improve the iron overload observed in thalassemia intermedia patients without any noticeable side effects. Placenta-derived extract Laennec and Porcine are effective drugs in this type of iron overload disorder. In Egypt, β-thalassemia is the most common genetically determined, chronic, hemolytic anemia, with an estimated carrier rate of 9–10.5% [72]. Currently, thalassemias are managed with chelation therapy, but exogenous Tf [73], exogenous hepcidin [74], or hepcidin signaling agonists [75] may be used as effective options in the near future. Our findings on the efficacy of Laennec and Porcine will help in the appropriate management of not only HH patients but also thalassemia (intermedia, trait, and major) and MDS patients.

This study is the first to present the possibility of applying the placenta-derived drugs Laennec and Porcine for the control of preferable and desirable erythropoiesis. β-thalassemia causes ineffective erythropoiesis and chronic anemia and is associated with iron overload from both multiple blood transfusions and increased iron absorption; the latter is mediated by suppression of the iron-regulatory hormone hepcidin [76]. In accordance with these backgrounds, we should seek to determine whether β-thalassemia major/intermediate/trait, with or without transfusion-mediated inhibition of erythropoiesis, will improve after treatment with Laennec and Porcine as a hepcidin inducer.