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http://www.kidney-international.org & 2014 International Society of Nephrology

Emerging treatments for amyloidosis Rabya H. Sayed1,2, Philip N. Hawkins1 and Helen J. Lachmann1,2 1

National Amyloidosis Centre, UCL Medical School, Royal Free Hospital, London, UK and 2UCL Centre for Nephrology, UCL Medical School, Royal Free Hospital, London, UK

Amyloidosis results from protein misfolding, and ongoing amyloid deposition can ultimately lead to organ failure and death. Historically, this is a group of diseases with limited treatment options and frequently poor prognosis. However, there are now ‘targeted’ therapeutics emerging in the form of stabilizers of the precursor protein, inhibitors of fibrillogenesis, fibril disruptors, and blockers of protein translation, transcription, and immunotherapy. We review many of these approaches that are currently being assessed in clinical trials. Kidney International advance online publication, 3 December 2014; doi:10.1038/ki.2014.368 KEYWORDS: amyloidosis; chemotherapy; diflunisal; eprodisate; immunotherapy; oligonucleotides

The term amyloidosis encompasses a group of diseases caused by the pathogenic misfolding of specific proteins that differ substantially with respect to organ involvement, management, and prognosis. Recent diagnostic developments have improved disease recognition, and the incidence of systemic amyloidosis is estimated to be at least c. 8 per million per year, although likely to be higher.1 Immunoglobulin lightchain associated (AL) and secondary (amyloid A (AA)) amyloidoses are by far the most prevalent subtypes with renal involvement present in two-thirds and 490% of patients, respectively.2,3 Renal presentation mainly reflects glomerular deposits with proteinuria, often overt nephrotic syndrome, and varying renal insufficiency.4,5 However, where tubulointerstitial involvement predominates, particularly in some types of hereditary apolipoprotein A-1 amyloidosis, a gradual decline in renal excretory function, without significant proteinuria, occurs.6 Untreated, median survival with AL amyloidosis is only 12 months; however, survival after treatment now exceeds 3 years,7 and it remains critically important that we strive to further ameliorate both the morbidity and mortality burden associated with systemic amyloidosis. This review specifically focuses on the novel therapies that have lately entered the clinical testing arena or that loom on the horizon. PATHOGENESIS

Correspondence: Helen J. Lachmann, National Amyloidosis Centre, UCL Medical School, Royal Free Hospital, Rowland Hill Street, London NW3 2PF, UK. E-mail: [email protected] Received 28 February 2014; revised 22 April 2014; accepted 24 April 2014 Kidney International

The amyloidoses are the consequence of protein misfolding and aggregation, subsequent insoluble fibril formation, and accumulation in the extracellular space, ultimately causing organ dysfunction and death.8 More than 30 different amyloid fibril precursor proteins are known in humans, but all fibril types essentially share a similar ultrastructure.9 The acquired highly characteristic b-pleated conformation of amyloid fibrils is associated with specific biophysical properties, including the ability to bind Congo red dye in a spatially ordered manner that produces diagnostic green birefringence when viewed under cross-polarized light10,11 (Figure 1). Under electron microscopy, amyloid deposits appear as randomly arranged, rigid nonbranching fibrils of B10 nm in diameter and of indeterminate length.12 The mechanisms by which amyloid deposits cause tissue damage have not yet been fully elucidated. The presence of large amounts of amyloid material can disrupt tissue architecture and mechanically interfere with the physiologic function of affected organs.13 Prefibrillar oligomeric species 1

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RH Sayed et al.: Emerging treatments for amyloidosis

a

b

c

Figure 1 | Sections of a renal biopsy sample were stained with Congo red dye and viewed at 200 magnification. (a) Amorphous deposits of eosinophilic material are visible within the glomerulus. (b) Pathognomonic green birefringence of amyloid deposits is visible when viewed under cross-polarized light. (c) Immunostaining of the amyloid deposits with anti-k antibodies was strongly positive (brown stain).

Table 1 | The major amyloid subtypes Amyloid subtype

Fibril precursor

Treatment

Clinical involvement

AL

Monoclonal-free immunoglobulin light chains

AA

Serum amyloid A protein

Renal (50–80%), cardiac, liver, spleen, bones, GI, autonomic and peripheral neuropathy, soft tissue Renal (495%), liver, spleen, adrenals, autonomic neuropathy

ATTR: wild-type ATTR: hereditary

Wild-type transthyretin Variant transthyretin

b2M

b2-Microglobulin (associated with chronic dialysis)

AFib (hereditary)

Variant fibrinogen Aa

Apolipoprotein AI (hereditary) Apolipoprotein AII (hereditary) Lysozyme (hereditary)

Variant apolipoprotein AI

Chemotherapy directed at the underlying plasma cell dyscrasia Potentially novel agents Treatment aimed at the specific underlying inflammatory condition Potentially eprodisate Mainly supportive with optimization of fluid status Cardiac transplantation Potentially novel agents Liver±cardiac±renal transplantation Tafamadis Potentially novel agents Mainly supportive, for example, splints, braces, collars. High-flux dialyzer membranes, frequent hemodialysis, b2M adsorption columns to reduce formation Renal transplantation Renal±liver transplantation Potentially novel agents Renal þ /  liver transplantation Potentially novel agents Renal transplantation Potentially novel agents Renal transplantation Potentially novel agents

Variant apolipoprotein AII Variant lysozyme

Cardiac, soft tissue Dominant neurological±cardiac involvement (dependent upon specific TTR variant)

Osteoarticular, bone cysts, soft tissue. Late visceral deposition including cardiac, GI, and spleen Renal Renal (mainly medullary), liver, heart, skin, larynx Renal Renal, liver, GI, spleen, lymph nodes, lung, thyroid, salivary glands

Abbreviations: AA, amyloid A; AFib, fibrinogen Aa-chain; AL, immunoglobulin light chain amyloidosis; ATTR, amyoidogenic transthyretin; b2M, b2-microglobulin; GI, gastrointestinal; TTR, transthyretin.

may also be toxic and contribute to organ dysfunction; cytotoxicity, in these cases, seems to be related to structural flexibility and exposure of hydrophobic residues.14 The major types of systemic amyloidosis are described in Table 1. Ideally, immunofluorescence on fresh tissue or, failing that, immunohistochemistry on fixed sections should be used to distinguish between the different types.12 Sometimes a characteristic distribution of deposits on light microscopy alone can provide diagnostic pointers––for example, isolated heavy glomerular involvement in fibrinogen Aa amyloidosis.15 Genetic testing is invaluable in diagnosing and excluding the known hereditary forms of amyloidosis, and fibril typing by mass spectrometry is increasingly used when immunostaining fails to provide definitive results.16 TRANSTHYRETIN AMYLOIDOSIS (ATTR)

Transthyretin (TTR) is a 55-kDa homotetrameric plasma protein that transports thyroxine and Vitamin A and is associated in its wild-type (wt) form with acquired amyloidosis, 2

termed wild-type TTR (wtTTR) amyloidosis and formerly known as senile systemic amyloidosis.17,18 More than 100 genetic variants of TTR are associated with autosomal dominant hereditary amyloidosis, and these usually involve the peripheral and autonomic nervous system and/or the heart.19,20 Notable variants include TTR Val30Met, which is the most common cause of familial amyloid polyneuropathy (FAP), and Val122Ile, which occurs in B4% of African Americans and is associated with late-onset familial amyloid cardiomyopathy, although with quite low penetrance.21–25 Untreated FAP is a progressive disease resulting in death within 7–15 years;26 although renal amyloid deposits occur, only 34.6% develop chronic kidney disease and 10% progress to end-stage renal failure.27 The conversion of circulating TTR protein into amyloid requires dissociation of the normal tetrameric protein into monomers, conformational change, and assembly to form the fibrils.19,21 The propensity to form amyloid is influenced by specific amino-acid substitutions and environmental factors such as pH and oxidative stress.28 Kidney International

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RH Sayed et al.: Emerging treatments for amyloidosis

WtTTR amyloidosis is very likely to be an underdiagnosed condition that commonly presents in the older population with symptoms predominantly of right-sided heart failure and often a history of the carpal tunnel syndrome.29 Supportive treatment is with diuretics, antiarrhythmics or pacemaker implantation, anticoagulation where supraventricular arrhythmias are present, and an avoidance of digoxin and calcium channel blockers. Antihypertensives are usually poorly tolerated as these patients can be profoundly hypotensive.30,31 Circulating TTR protein is almost exclusively liver derived,32 and the ‘surgical gene therapy’ with liver transplantation has been undertaken in more than 2000 FAP patients since 1990.33,34 Liver transplantation, when performed early, prolongs life, particularly in patients with the TTR Met30 variant, but neurological impairment and a poor quality of life remain problematic.35 In other mutations, and in older patients, outcomes are less favorable, and may reflect postoperative amyloid cardiomyopathy.36 This is thought to be due to the deposition of wt TTR on a template of preexisting cardiac amyloid derived from variant proteins.37 The need for better therapies has resulted in the development of a number of novel strategies, and some of these have been assessed in clinical trials. STABILIZATION OF THE TTR TETRAMER Diflunisal

Diflunisal is a nonsteroidal anti-inflammatory drug that stabilizes tetrameric TTR in vitro by binding via the thyroid hormone receptor sites.38,39 Berk et al.40 recently completed an international, multicenter, placebo-controlled study of 130 FAP patients, with neurological outcome as the primary end point. The exclusion criteria included patients 475 years of age, a history of gastrointestinal bleeding, estimated glomerular filtration rate o30 ml/min, and NYHA (New York Heart Association) Class IV heart failure. Sixty-three patients with a median age of 59.7 years, of whom 54.6% had TTR Met30, completed the study, having taken either 250 mg diflunisal or placebo daily over a 2-year period. The authors reported a significant reduction in the rate of progression of neurological impairment and an improved quality of life in the diflunisal-treated group, with 29.7% of the diflunisal and 9.4% of the placebo group displaying a stable neurological score by Neuropathy Impairment Score plus 7 nerve tests (NIS þ 7) at 2 years. Drug-related adverse events, including gastrointestinal bleeding and renal dysfunction, did not differ between the two groups.40

undertook a randomized phase II/III double-blinded trial that assigned either 20 mg oral tafamadis or placebo to 128 FAP patients (mean age 39 years) with the V30M TTR mutation and early neuropathy for a period of 18 months. Plasma TTR stabilization was seen in 98% of tafamadistreated patients and none of the placebo arm. The study did not show a significant change in the predefined primary end point but suggested a slowed deterioration in small fiber neuropathy in the tafamadis arm at 18 months, with 45.3% of the evaluable diflunisal and 29.5% of the placebo group displaying a stable neurological score by NIS of the lower limb at 18 months. The most common side effects included urinary tract infections and diarrhea. There has been no evidence that Tafamadis affects thyroid function tests; however, routine monitoring is advised. Notably, Coelho’s study excluded patients with an estimated glomerular filtration rate of o30 ml/min, and there currently remains no evidence of efficacy in cardiac TTR amyloidosis. This study has split the drug regulatory authorities with the European Medicine Agency granting approval for Tafamadis use in FAP for TTR Met30 patients with early disease who can still walk independently. It has not yet been approved by the US Food and Drug Administration. These two studies highlight some of the difficulties in conducting drug trials in amyloidosis and amyloid neuropathy in particular. These include problems with patient selection (the study populations were very different) and quantifying the amyloid deposits; there are no imaging techniques that can accurately identify let alone quantify neural amyloid deposits, and biopsies are subject to sampling error. In addition, nerve biopsies can only be used very sparingly, and it is not clear that assessment of amyloid by serial fat aspirates (a safe and easily obtained source of tissue) provides a reliable insight into changes in amyloid load. The assessment of neurological function is currently onerous and dependent on clinical experience and subjective interpretation. The widely used measures reported in these two studies are taken from the diabetic literature and may not be entirely appropriate; the rate of deterioration of neurological function in FAP is much more rapid than that seen in diabetes, and there have been no prospective observational studies to validate these methodologies in amyloid neuropathy. The numerical scores that are generated give the impression that any particular change in score has similar clinical weight regardless of baseline function, and this seems inherently unlikely. Ongoing questions relating to whether either of these agents will be beneficial in cardiac amyloidosis are being addressed at present.

Tafamadis

Tafamadis (2-(3,5–dichloro–phenyl)-benzox-azole-6-carboxylic acid) is an orally administered drug that acts to stabilize the TTR tetramer through its affinity for the T4binding site, and it does not carry the risks associated with nonsteroidal anti-inflammatory drug use.41 It is protein bound, metabolically stable, and X94% is excreted unchanged from the digestive tract. Coelho et al.42 Kidney International

Epigallocathechin-3-gallate

Recent in vitro experiments show that 50 mmol/l epigallocatechin-3-gallate, the most abundant catechin in green tea (GT), efficiently inhibits fibril formation from amyloid b-protein, a-synucleine, and TTR and converts existing fibrils into nonfibril conformers.43,44 TTR tetramer stabilization is observed when epigallocathechin-3-gallate binds to 3

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RH Sayed et al.: Emerging treatments for amyloidosis

recombinant wt and variant TTR tetramers at three sites different from the thyroxine-binding site.45 Kristen et al.46 undertook a prospective study of 19 patients with cardiac TTR amyloidosis consuming either standardized GT extract in the form of capsules or 1.2–2 l of GT/day over a year. Ten patients had hereditary ATTR amyloidosis and 9 patients had wtTTR amyloidosis. Five patients did not complete the study (2 died, 2 discontinued GT/GT extract, and one underwent heart transplantation). In the subgroup of patients evaluated by cardiac magnetic resonance imaging, a mean decrease in 12.5% left ventricular mass was detected. There were no serious adverse effects reported by any of the participants.46

RNA polymerase 5′

Mello,47

The discovery of RNA interference by Fire and for which they were awarded the Nobel Prize in Physiology in 2006, demonstrated how the transfer of genetic information from DNA to protein can be blocked. Oligonucleotide-based therapies, including small interfering RNAs (siRNAs) and antisense RNAs, have the ability to cause changes at the translational level without becoming integrated into the human genome.48 The siRNAs are noncoding, double-stranded molecules that are components of the endogenous RNA interference pathway that serves to control gene expression. They vary in length from 18 to 30 base pairs and are chemically modified for drug delivery to increase stability and limit immunogenicity.49 Lipid nanoparticles have been used to deliver siRNAs to hepatocytes parenterally; these have resulted in a robust and durable reduction in genetic expression across a variety of species.50 Antisense oligonucleotides (ASOs) are 13–25 nucleotide single-stranded DNA molecules that hybridize to a specific messenger RNA (mRNA) sequence and prevent transcription.51,52 The mechanisms through which both types of oligonucleotide work are illustrated in Figure 2. Oligonucleotide therapies for ATTR have shown great promise in preclinical models, and both siRNAs and antisense RNAs are being investigated in clinical studies (ClinicalTrial.gov NCT01960348 and NCT01737398). Coelho et al.53 undertook a single-dose, randomized, placebo-controlled phase I trial using ALN-TTR01 and ALN-TTR02 (firstand second-generation lipid nanoparticles); these contain an identical siRNA that binds to an mRNA segment common to both wt and mutant TTR. There was a rapid, dose-dependent, and durable reduction in transthyretin levels in the 32 patients with TTR amyloidosis.53 Alnylam Pharmaceuticals (Massachusetts) have developed an RNA interference therapeutic that has been chemically modified by conjugation to an Nacetylgalactosamine moiety, thereby refining hepatocyte targeting and allowing subcutaneous administration;49,54,55 this is currently in phase II trials for cardiac ATTR amyloidosis. ASOs have already been tested clinically in the treatment of viral diseases, cancer, and metabolic disease.56–58

Nucleus

Rewinding of DNA

3′ mRNA 5′

3′ Promoter region

3′

Unwinding of DNA . .

1

5′

siRNA

2 Antisense oligonucleotide

Oligonucleotide-based therapies

4

Hepatocyte

siRNA–RISC complex

TTR

3

5 4

Cytoplasm

Figure 2 | Hepatocyte schematic demonstrating the mechanism by which small interfering RNAs (siRNAs) block the transcription process and antisense oligonucleotides interfere with the translation process, ultimately preventing transthyretin (TTR) formation. (1) The siRNAs bind to the RNA-inducing silencing complex (RISC) in an adenosine triphosphate (ATP)–dependent manner. (2) This multisubunit protein complex migrates toward messenger RNA (mRNA). At some point, the siRNA unwinds and the antisense strand remains bound to the RISC and blocks transcription by the direct degradation of the target mRNA sequence through the use of both endo- and exonucleases.37, 42 (3) The mRNA migrates into the cytoplasm where (4) hybridization with the antisense oligonucleotide prevents protein translocation (5).

ISIS-TTRRX is a second-generation chimeric antisense inhibitor of TTR. It binds selectively and with high affinity to the nontranslated portion of the human TTR mRNA and results in its degradation, preventing production of both wt and variant TTR protein. ISIS-TTRRX twice-weekly subcutaneous injections have been well tolerated in both TTR transgenic mouse models and monkeys with a reduction in hepatic TTR mRNA and plasma wt TTR protein levels by B80%, and it is currently under evaluation in a phase I clinical trial in normal healthy volunteers.26,59 It will initially be developed for patients with FAP, and a randomized, double-blind, placebo-controlled study to assess the longterm safety and efficacy of ISIS-TTRRx in patients with FAP was initiated in 2013. The mechanism by which ASOs mediate their effect could be beneficial in other types of amyloidosis. Kluve-Beckerman et al.60 used ASOs to suppress serum amyloid A protein (SAA) production in mice and demonstrated that SAA levels in ASO-treated mice were 63% lower than those in controls, resulting in reduced AA amyloid deposition. Kidney International

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SYSTEMIC AA AMYLOIDOSIS

Systemic AA amyloidosis results in organ dysfunction due to extracellular deposition of N-terminal fragments of SAA in the form of insoluble amyloid fibrils. These deposits have a predilection for the kidneys, spleen, liver, and intestines, with the kidney being the most commonly affected organ. Patients typically present with progressive proteinuria, nephrotic syndrome, and renal insufficiency.5,61 SAA is a hepatic acutephase response protein, and synthesis can be upregulated by 1000-fold in response to inflammatory cytokines, particularly interleukins 6 and 1, and, to a lesser extent, tumor necrosis factor-a.62–64 A persistent inflammatory response can arise from any variety of causes, the more common ones being rheumatoid arthritis, seronegative arthritides, inflammatory bowel disease, and long-standing infections such as osteomyelitis, decubitis ulcers, and bronchiectasis.61 It is not yet known what predisposes certain patients to develop systemic AA amyloidosis; there has been no one specific gene or allele identified, and it may be that the frequency and severity of inflammatory episodes and thus the degree and duration of elevated SAA levels is the primary risk factor. Other identified risk factors include the SAA1 isotype and perhaps variants in metalloproteinases.65 Renal transplantation

Renal involvement is the most frequent and serious complication of AA amyloidosis,66 necessitating the need for renal replacement therapy including renal transplantation. Pinney et al.67 described 43 patients with AA amyloidosis with renal transplants and a median follow-up of 5.1 years. Graft loss due to recurrent amyloid occurred in only 4.6% of cases and predicted median graft survival was 10.3 years. Lower SAA levels were associated with an improved outcome, with graft survival, noncensored for death, of 14.5 years in patients with a median SAA value of o10 mg/l and 7.8 years in those with a median SAA value of 410 mg/l. Furthermore, the median SAA concentration was significantly higher among those with graft amyloid recurrence. Of the patients, 37% died, with approximately two-fifths of deaths being due to infection. Kofman et al.68 showed that their cohort of 59 transplanted AA patients had a 5-year patient survival of 83.1% and graft survival (censored for death) of 83.1%. Patient survival was less good compared with the outcome of 179 age-matched renal transplant recipients, although graft survival was not statistically different.68 A total of 72.3% developed at least one infection, and 43% of patient deaths were due to severe sepsis. They postulated that this risk arose from both preand post-transplant immunosuppression. Current drug treatment

Current treatment is aimed at reducing SAA production to healthy normal levels (3 mg/l) through control of the respective underlying inflammatory disease. Sustained suppression of SAA production results in amyloid regression.69 Lachmann et al.61 reported an improvement in renal functions in patients with a median SAA concentration of 6 mg/l and Kidney International

deterioration in patients with a median SAA concentration of 28 mg/l. Proteinuria may diminish substantially, although gradually over months and even years, in patients with AA amyloidosis when the underlying inflammatory disease remains quiescent.61 Eprodisate

Glycosaminoglycans, such as heparin sulfate, promote fibril assembly by acting as chaperones during early stages of protein refolding and amyloid formation. Eprodisate (Kiacta, Neurochem) is a negatively charged, sulfonated molecule that is structurally similar to heparin sulfate and works by competitively inhibiting the interaction between SAA and glycosaminoglycans. It has inhibited AA amyloid development in experimental mouse models. A multicenter, randomized, double-blind, placebo-controlled trial in 180 patients with AA amyloidosis–associated nephropathy was completed.70 As eprodisate is renally cleared, the dosage of daily drug prescribed was dependent upon creatinine clearance. Study medication was continued for 24 months, unless the patient progressed to end-stage renal disease, had a significant adverse event, withdrew from the study, or required rescue medication such as cytotoxic agents, colchicine, or anti-tumor necrosis factor agents. Rheumatoid arthritis (49% of the patients) and familial Mediterranean fever (19%) were the most common underlying inflammatory diseases. Treatment with eprodisate was associated with a 42% reduction in the risk of worsening renal disease (as measured by creatinine clearance) or death (0.37–0.93 P ¼ 0.02). More specifically, compared with placebo, eprodisate significantly reduced the risk of doubling of serum creatinine, the risk of a 50% reduction in creatinine clearance, and the slope of decline in creatinine clearance. The decline in creatinine clearance was 4.7 ml per min per 1.73 m2 per year greater in the placebo group than in the eprosidate group, a relative difference of 30%.70 Perhaps surprisingly, there was no significant difference in terms of the overall changes in proteinuria. Despite these encouraging results, there was some concern that some of the apparent drug benefit reflected a poor outcome in the subgroup of placebo patients who had nephrotic-range proteinuria at baseline, and the US and European regulatory bodies requested a confirmatory second phase III trial that is ongoing.71 AL AMYLOIDOSIS

Systemic AL amyloidosis is by far the most frequently recognized type of amyloidosis with potentially significant multiorgan involvement. Fibrils are derived from monoclonal immunoglobulin light chains, and AL amyloidosis most commonly occurs in patients with otherwise asymptomatic and low-grade clonal plasma cell dyscrasias, although 10–15% of patients have multiple myeloma.12,72 Current treatment centers on suppressing clonal B cells, and hence reducing the supply of the amyloidogenic fibril precursor protein. This may facilitate gradual regression of amyloid deposits and stabilization or improvement in vital organ function (Figure 3). There is convincing evidence that more complete 5

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Table 2 | Mayo cardiac amyloid staging system Variable cTNT NT-proBNP Median survival (months)

Stage I

Stage II

Stage III

o0.035 mg/l

X0.035 mg/l or X332 ng/l 10.5

X0.035 mg/l

o332 ng/l 26.4

X332 ng/l 3.5

Abbreviations: cTNT cardiac troponin T; NT-proBNP, N-terminal pro-brain natriuretic peptide. This staging system is widely used in studies when evaluating therapeutic efficacy. Higher stages correlate with a worse prognosis.64

Figure 3 | Iodine-123 (123I)-labeled serum amyloid P (SAP) component scintigraphy. Anterior-posterior view of an SAP scan, demonstrating (a) a large total body amyloid load with hepatic and splenic involvement (b) with regression after nine cycles of chemotherapy, resulting in a small total body amyloid load.

clonal responses are associated with both longer treatmentfree survival and a greater chance of improvement in organ function. In low-risk patients, autologous stem cell transplantation is widely regarded as the treatment of choice, although this was not supported by the only randomized study to be performed,73 and outcomes depend heavily on patient selection and center experience. The prognosis of untreated AL patients remains 12–15 months,74 and just a few months for patients with significant cardiac involvement, highlighting the need for novel, more effective regimens. The pathological light chains may have a direct cardiotoxic effect and, although renal failure clearly adds to disease burden, it is actually cardiac damage that is the major prognostic marker.75 This is assessed by the Mayo staging system (Table 2) and improvement after treatment directly affects prognosis.76 However, one should bear in mind the effect of a diminishing renal excretory function on the widely used prognostic markers: serum cardiac troponin T and the NT-proBNP (N-terminal fragment of pro-brain natriuretic peptide). Importantly, as organ response can often be delayed, treatment efficacy is monitored through the measurement of hematological parameters, specifically serum-free light-chain measurements (Table 3).77 Current treatment

The agents currently used include the newer immunomodulatory drugs and the proteasome inhibitor, bortezomib, as well as the more traditional regimes (Table 4). Current regimes have been modified from multiple myeloma protocols, and treatment choice depends upon the type and severity of organ involvement––for example, cardiac involvement, peripheral neuropathy, or significant hypotension may preclude particular agents. These drugs are commonly combined with 6

dexamethasone and are often used in conjunction with a myelosuppressive agent, such as cyclophosphamide. Melphalan and dexamethasone have been used to reduce the production of aberrant light chains, and although 33% of ‘intermediate-risk’ patients achieved complete response (CR) with high-dose dexamethasone and a median survival of 5.1 years, CRs were halved in patients with significant cardiac AL (who could only tolerate low-dose dexamethasone), 26% of whom died during the period of treatment.78,79 Furthermore, there is significant myelosuppression associated with intermediate-dose melphalan.80 The potential advantage of autologous stem cell transplantation (ASCT) in delivering a longer remission-free period and a relatively quick CR) means that patients should be considered for this procedure from the onset. The depth of hematological response is associated with the degree of organ response, emphasizing the importance of trying to achieve CR either through chemotherapy alone or with ASCT. Autologous stem cell transplantation

This was first reported as a major breakthrough in AL amyloidosis by Comenzo et al.81 in 1998. Recently, Cibeira et al.82 showed that a CR, after ASCT, confers an overall survival of 86% at 5 years and an event-free survival of 8.3 years compared with 2 years for patients not in CR. However, treatment-related mortality has been reported as being as high as 40% in 1999, and since then efforts have been made to risk stratify patients more rigorously.83 Gertz et al.84 undertook the largest study of patients receiving ASCT and have recently refined their eligibility criteria, as analysis showed that higher levels of cardiac biomarkers were the sole predictors of early mortality after ASCT, and treatment-related mortality was o1% if the NT-proBNP was o5000 pg/ml and troponin was o0.06 ng/ml. In selected low–intermediate-risk AL patients, ASCT is well established as a first-line treatment, but the use of stringent criteria, designed to reduce treatment-related mortality, means that only 14% of newly diagnosed cases seen in the UK national center have been considered for ASCT. Immunomodulatory drugs

Thalidomide is most commonly combined with cyclophosphamide and dexamethasone.85,86 Of the patients, 33% have been reported to have achieved a complete or very good Kidney International

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Table 3 | Current international criteria for monitoring hematological response Response category

Criteria

Complete response Very good partial response Partial response No response

Normalization of FLC levels and ratio, negative serum and urine immunofixation Reduction in the dFLC to o40 mg/l 450% reduction in the dFLC o50% response in dFLC

Abbreviations: dFLC, difference between the involved FLC and the uninvolved FLC; FLC, free light chains.66

Table 4 | Current chemotherapy regimens for AL amyloidosis Family

Primary agent plus adjunctive treatment

Alkylator

Melphalan–dexamethasone

Immunomodulatory

Thalidomide (with cyclophosphamide and dexamethasone)

Immunomodulatory (second generation)

Lenalidomide (with dexamethasone)

Immunomudulatory (third generation)

Pomalidomide (with dexamethasone)

Alkylator and purine analog Proteasome inhibitor

Bendamustine (and dexamethasone) Bortezomib (and dexamethasone± cyclophosphamide or with dexamethasone and melphalan)

Main side effects

Comments

Hematological toxicity Fatigue Peripheral edema GI side effects Fluid retention Fatigue and postural hypotension Peripheral neuropathy Thromboembolism Increase in cardiac biomarkers Skin rash Teratogen Fatigue Constipation/diarrhea Myelosuppression Thromboembolism Skin rashes Increase in cardiac biomarkers Renal dysfunction Fluid retention Fatigue and postural hypotension Peripheral neuropathy Thromboembolism Myelosuppression Increase in cardiac biomarkers Skin rash Teratogen Cytopenias

Good choice for intermediate-risk and frail patients without significant cardiac involvement. IV MDex too toxic for routine use but used if poor GI absorption

Peripheral neuropathy Hypotension GI disturbance Peripheral edema

Useful in disease refractory to alkylators and bortezomib. Response is not achieved rapidly. Addition of cyclophosphamide or melphalan has improved the CR rate but two-thirds of patients develop side effects

Useful where disease is relapsed/refractory to lenalidomide and thalidomide including cardiac patients

Useful for relapsed/refractory disease Advised upfront in those with a poor prognosis where a rapid response is required

Abbreviations: AL, immunoglobulin light chain amyloidosis; CR, complete response; GI, gastrointestinal; IV MDex, intravenous melphalan-dexamethasone.

partial response, after a median of 7 months, but 29% of patients died and 50% of treated patients had to be hospitalized for treatment toxicities, mainly fluid retention, lethargy, infection, hypotension, and neuropathy.87 Lenalidomide has a hematological response rate ranging from 41 to 47%, including relapsed and thalidomide refractory cases. Doses higher than 15 mg are poorly tolerated with side effects including cytopenias, fatigue, and fluid retention. In a retrospective analysis from the Boston group, 66% of patients exposed to lenalidomide developed renal dysfunction, which was reversible in 44% of cases.88 However, it was reasonably well tolerated in patients with end-stage renal failure. There has also been an observed increase in NT-proBNP and other cardiac biomarkers with the use of immunomodulatory agents, and this has been Kidney International

correlated with mortality.89 Time to hematological response is longer than that observed with bortezomib, but CRs have been seen.90 Pomalidomide

This thalidomide analog was reported by Dispenzieri et al.91 to induce a 48% hematological response rate with 3% CRs in 33 heavily pretreated patients. The 1-year progression-free survival and overall survival rates were 59% and 76%, respectively. Of the 33 patients, 12 had renal involvement, and 2 of these patients demonstrated organ improvement.91 Proteasome inhibitors

Bortezomib induces a rapid decrease in serum-free lightchain concentration in patients with myeloma.92 Purified 7

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plasma cells from amyloid patients are twice as vulnerable to bortezomib inhibition as those from myeloma patients.93 This is thought to be because amyloidogenic light chains have a greater propensity to misfold, thus overloading the proteasome. Its efficacy in achieving both a high hematological and organ response rate has led to it being adopted as a frontline therapy in AL amyloidosis,94 and it is being increasingly used in those with severe cardiac involvement whose outlook is extremely poor.95,96 Venner et al.97 reported a hematological response of 81.4% using biweekly bortezomib, cyclophosphamide, and dexamethasone, superseding that achieved by other combinations including 71% with bortezomib/ dexamethasone,98 67% with bortezomib/ melphalan/dexamethasone,99 and 69% with bortezomib alone.100 Other novel proteasome inhibitors currently undergoing trials include ixazomib and carfilzomib.94,101 GENERIC ANTI-AMYLOID APPROACHES

Although known treatments such as chemotherapy in AL type and anti-inflammatory drugs in AA type can halt and/or slow down the production of amyloid fibrils, these do not work to promote the clearance of existing deposits that can be a cause of considerable morbidity and, in some instances, organ and patient death. Immunotherapy

Using passive immunotherapy to produce a regulated immune response against amyloid deposits is an attractive approach to increase amyloid clearance. Such antibodies could be directed against specific amyloid proteins such as immunoglobulin light chains or SAA or against constituents that are common to all types of amyloidosis such as serum amyloid P component (SAP). Wall et al.102 have generated fibril-specific monoclonal antibodies that have been shown to opsonize and promote clearance of AL and AA amyloid in mice models. Building on this work, a company called Prothena has been granted orphan drug status for their monoclonal antibody NEOD001 that has been designed to specifically target AL amyloidosis. A phase I clinical trial investigating safety and tolerability in patients with systemic AL in 2013 is now open for recruitment in seven US centers (ClinicalTrials.gov Identifier: NCT01707264). CPHPC and anti-human SAP antibodies

SAP, a normal nonfibrillar plasma glycoprotein synthesized by hepatocytes, is a constituent of all amyloid deposits and can comprise up to 14% of the dry mass of amyloid. SAP binding both physically stabilizes amyloid fibrils103 and protects them from proteolysis and degradation by phagocytic cells. R-1-[6-[R-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl] pyrrolidine-2-carboxylic acid (CPHPC) is a competitive inhibitor of SAP binding to amyloid fibrils. It consists of two D-proline residues joined by a six-carbon aliphatic linker. The palindromic structure of this drug crosslinks pairs of SAP molecules in the plasma that triggers their complete clearance by the liver, eventually gradually depleting SAP from the amyloid depos8

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its.104,105 It also rapidly depletes SAP from the CSF. Gillmore et al.74 undertook a prospective study in 31 patients with advanced systemic amyloidosis of AL, fibrinogen Aa-chain, ATTR, Gelsolin, ApoA1, AA, and Ab2M types. The patients received CPHPC by twice-daily subcutaneous injection, and six subjects continued to receive CPHPC for another year. This treatment produced a sustained and profound depletion of circulating SAP in all cases, and in the two patients in whom amyloidotic material became available for analysis there was also substantial SAP depletion from the amyloid deposits. In four of the five dialysis-independent patients with fibrinogen Aa-chain amyloidosis, proteinuria decreased, whereas proteinuria increased and renal survival was shorter in five of six matched untreated historical controls. Among 13 patients with advanced AL amyloidosis, organ function improved in 2 cases and was stable in 7 cases. In two AL cases with refractory clones and a third case with a modest clonal response, organ function deteriorated. The only adverse events attributable to CPHPC were transient minor local stinging at the injection site for two patients. There were five ‘expected’ deaths associated with advanced amyloid cardiomyopathy.74 Administration of anti-human SAP antibodies to mice with amyloid deposits containing human SAP triggers a potent, complement-dependent, macrophage-derived giant cell reaction that swiftly removes massive visceral amyloid deposits without adverse effects. As short-term treatment with CPHPC almost completely depletes SAP from the circulation but not from amyloid deposits, passively administered anti-SAP antibodies can specifically target amyloid deposits. A study with amyloidotic human SAP transgenic mice was undertaken comprising pretreatment with CPHPC for 5 days followed by intraperitoneal injection of either an IgG fraction of monospecific polyclonal sheep anti-human SAP serum or a control sheep IgG or no injection. The visceral amyloid load was scored histologically 28 days later. The amyloid deposits in mice treated with CPHPC and anti-SAP antibodies were massively reduced compared with those having received CPHPC alone or with control antibodies. There was no disruption to the normal structure of the affected viscera, and there were no clinical or biochemical adverse effects. Histological analyses at 24 h after anti-SAP antibody administration showed that all deposits were densely infiltrated with mononuclear inflammatory cells and some granulocytes. On day 2, macrophages fused to form giant cells and demonstrated phagocytic endocytotic activity. By day 16, amyloid clearance was largely complete. This process depends critically on macrophage activity, and ablation of macrophage activity using liposomal clodronate resulted in no reduction in amyloid load.106 Currently, a phase I/II trial is in progress to evaluate the effects and safety of co-administrating CPHPC with anti-SAP antibodies (Clinicaltrials.gov Identifier: NCT01777243). Fibril disruptors

Physical disruption of the structure of amyloid fibrils by small intercalating molecules may sufficiently destabilize the Kidney International

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fibrils, hence increasing their degradation. Gianni et al.107 in 1995 made the serendipitous observation that the administration of the anticancer drug 4’iodo-4’deoxyrubicin (I-DOX) to a patient with AL amyloidosis and multiple myeloma was associated with a rapid and impressive clinical improvement; this prompted further evaluation in eight patients with refractory AL amyloid. There was clinical improvement in five of the patients, and in two patients the improvement was immediate. I-DOX induced amyloid reabsorption in three patients with a measurable reduction in the interventricular septal thickness, massive urinary excretion of amyloid fragments, and substantial reduction in a large amyloidoma and in splenic and hepatic deposits. A further study by Merlini et al.108 found that I-DOX binds to amyloid fibrils, irrespective of amyloid subtype, and it does so in vitro and in vivo, possibly by inhibiting further growth of the amyloid fibrils and by promoting amyloid reabsorption. It has been proposed that I-DOX disrupts the fibrillar structure of the native fibrils, hence producing an intermediate structure that may be more readily available for enzymatic degradation.109 The resemblance of the polycyclic conjugated structure of the tetracyclines with a glycone moiety of iododoxorubicin, believed responsible for the antiamyloidogenic effect, has prompted further investigation into the less toxic tetracyclines. A phase II, open-label study is underway to investigate the effects of 100 mg b.d. of p.o. doxycycline and 250 mg t.d.s. of taurodesoxycholic acid in 20 patients with hereditary TTR, wtTTR amyloidosis, and a domino recipient. Two patients stopped treatment because of gastrointestinal side effects. The study so far has demonstrated stabilization of cardiac and neuropathic involvement in the remaining patients, although final results are awaited.110 CONCLUSION

Currently, treatment options in amyloidosis rely on reducing the supply of the precursor protein and thus depend absolutely upon accurate typing of the amyloid. There is no doubt that comprehensive disease staging and a tailored individualized approach improve patient outcomes, but treatment-related morbidity is a significant problem. This is especially so in AL amyloidosis where advanced cardiac disease is uniquely challenging. A further point in AL amyloidosis is that the underlying clonal disorder may be or become ‘refractory,’ thereby requiring a number of lines of therapy from a chemotherapeutic armamentarium. Advances in the understanding of protein misfolding and its pathogenic consequences have led to an unprecedented number of novel treatment approaches. Stabilization of the protein precursor by agents that bind and ‘lock’ them into their normal configuration such as Tafamadis and Diflunisal have emerged as agents to slow down the deterioration in TTR amyloid, a disease that previously could only be addressed by liver transplantation. Even more radically, siRNA and antisense oligonucleotides can target specific genes to ‘switch off ’ amyloid protein production. Eprodisate, through Kidney International

interference with early mechanisms in fibril formation, has shown promise in patients with renal AA amyloidosis. Once mature fibrils have been deposited, physical disruption by intercalating agents such as doxycycline may destabilize the amyloid, and early clinical studies suggest this may in turn stabilize organ function. An exciting new development has been the emergence of immunotherapies that have the unique potential to promote clearance of amyloid deposits. Human studies, currently being undertaken with both monoclonal anti-AL antibodies and anti-SAP antibodies, hope to replicate the promising results achieved in mouse models. This review of potential therapeutic options demonstrates how research in amyloidosis has embraced advances in molecular biology and has entered an exciting era where basic science has now been translated into human trials. DISCLOSURE

All the authors declared no competing interests. ACKNOWLEDGMENTS

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