A Constantinou

C Chen

M P Deonarain
email: m.deonarain@imperial.ac.uk

Á C Chen

Modulating the pharmacokinetics of therapeutic antibodies

Keywords: Conjugate, Fab, Fusion protein, Immunoglobulin, PEGylation, Pharmacokinetics, Polysialylation, scFv

With the advent of antibody fragments and alternative binding scaffolds, that are devoid of Fc-regions, strategies to increase the half-life of small proteins are becoming increasingly important. Currently, the established method is chemical PEGylation, but more elaborate approaches are being described such as polysialylation, amino acid polymers and albumin-binding derivatives. This article reviews the main strategies for pharmacokinetic enhancement, primarily chemical conjugates and recombinant fusions that increase apparent molecular weight or hydrodynamic radius or interact with serum albumin which itself has a long plasma half-life. We highlight the key chemical linkage methods that preserve antibody function and retain stability and look forward to the next generation of technologies which promise to make better quality pharmaceuticals with lower side effects. Although restricted to antibodies, all of the approaches covered can be applied to other biotherapeutics.

Introduction

The use of monoclonal antibodies (MAbs) as targeted therapeutic agents is now firmly established with many clinical and commercial successes [START_REF] Deonarain | Recombinant antibodies for cancer therapy[END_REF]. Many have become household names and in 2007, monoclonal antibodies became the largest class of commercial biotechnological agents [START_REF] Aggarwal | What's fueling the biotech engine-2007[END_REF].

Current MAbs used in the clinic fall largely into two categories: (1) cell-targeting antibodies which modulate receptor function and/or recruit effector functions; and (2) neutralizing agents for targets readily encountered in circulation (and to a lesser extent within tissues). However, the whole immunoglobulin format has limitations for some targeted applications. Their relatively large size makes them inefficient at penetrating deep tissue antigens [START_REF] Yokota | Microautoradiographic analysis of the normal organ distribution of radioiodinated single-chain Fv and other immunoglobulin forms[END_REF][START_REF] Graff | Theoretical analysis of antibody targeting of tumor spheroids: importance of dosage for penetration, and affinity for retention[END_REF][START_REF] Thurber | Factors determining antibody distribution in tumors[END_REF], while poor vascularisation within the tumor microenvironment can impede high dose antibody delivery [START_REF] Jain | Transport of molecules, particles, and cells in solid tumors[END_REF][START_REF] Fukumura | Imaging angiogenesis and the microenvironment[END_REF]. Further, in such cases where there is poor vascularization or if a patient is immunocompromised the Fc effector function of whole antibodies becomes largely redundant due to a limited supply of lymphocytes and macrophages (Badger et al. 1987). Moreover, the immunosuppressive microenvironment of tumors, as well as the absence of a complimentary Fc isotype, can impede appropriate Fc-directed functions [START_REF] Strome | A mechanistic perspective of monoclonal antibodies in cancer therapy beyond target-related effects[END_REF][START_REF] Dougan | The immune response to tumors[END_REF]. Another concern with the Fc domain is potential cross-reactivity with normal tissues, which can also lead to unwanted side effects, particularly when cytotoxinloaded antibodies are used. In order to address these characteristics, antibody formats, devoid of Fc-regions such as scFvs and Fabs have been engineered [START_REF] Holliger | Engineered antibody fragments and the rise of single domains[END_REF]. Being smaller, they are capable of more rapid penetration and clearance than whole mAbs.

When engineering antibodies for therapy, agents must generally exhibit high affinity, discriminating specificity, minimal immunogenicity and low crossreactivity [START_REF] Ober | Differences in promiscuity for antibody-FcRn interactions across species: implications for therapeutic antibodies[END_REF]. Normally these factors can be addressed by making relatively small changes to the primary protein structure of the antibody [START_REF] Carter | Potent antibody therapeutics by design[END_REF]. In addition to this, a major consideration in antibody engineering is developing one that exhibits optimal pharmacokinetics: appropriate dosing leading to optimal bioavailability, uptake, distribution and clearance in targeted and non-targeted tissues which will lead to optimal pharmacodynamics [START_REF] Beckman | Antibody constructs in cancer therapy: protein engineering strategies to improve exposure in solid tumors[END_REF].

There are two general approaches to extending the longevity and stability of therapeutic proteins: recombinant/genetic and chemical methods (Fig. 1). Chemical methods pre-date the recombinant approaches and involve the chemical attachment of moieties which alter the physico-chemical properties of the protein. Applied to antibodies, this includes the attachment of polymers, sugars and protein domains. Recombinant methods require the genetic engineering of the target protein or host cell line to produce variants with altered structure or function. The structural changes which lead to altered pharmacokinetics include size, hydrodynamic volume, aggregation status and electric charge (pI) and the functional changes include various receptor binding abilities. This review aims to survey the key technologies for pharmacokinetic modulation and will look forward to some derivatives which promise to become the next generation of antibody therapeutics. In the context of antibodies, pharmacokinetics can be affected by many parameters, including affinity for the target and valency. Modifications to the antigen binding site or multimerisation of antibodies are beyond the scope of this review. These topics are covered in excellent articles [START_REF] Pluckthun | New protein engineering approaches to multivalent and bispecific antibody fragments[END_REF][START_REF] Carter | Potent antibody therapeutics by design[END_REF][START_REF] Beckman | Antibody constructs in cancer therapy: protein engineering strategies to improve exposure in solid tumors[END_REF].

The most investigated method for modulating antibody pharmacokinetics has been by conjugation methods where chemically inert moieties are strategically attached. This has the effect of increasing the apparent size or hydrodynamic volume of a given particle so that its pharmacokinetics are significantly altered in relation to the conjugated size. The larger the conjugate attached the greater the half-life becomes. This is most significant at around the 50-70 kDa threshold which is the molecular weight cut-off for glomerular filtration [START_REF] Silbernagl | The renal handling of amino acids and oligopeptides[END_REF][START_REF] Akhtar | Molecular basis of proteinuria[END_REF]. This equates approximately to a diameter of around 90 A ˚for a globular protein. However, a balance must be achieved such that lower clearance rates are not gained at the expense of significantly poorer tissue penetration and/or poor tissue to blood ratios. This is a particularly important consideration if antibodies are used as carriers of cytotoxic agents, such as radioisotopes [START_REF] Davies | Radioimmunotherapy for B-cell lymphoma: Y90 ibritumomab tiuxetan and I(131) tositumomab[END_REF], where high doses leading to extended systemic residency could cause damage to normal tissues. However, for unconjugated antibodies (e.g. trastuzumab and rituximab) used to treat metastatic breast cancer and non Hodgkin's lymphoma respectively [START_REF] Hudis | Trastuzumab-mechanism of action and use in clinical practice[END_REF][START_REF] Bonavida | Rituximab-induced inhibition of antiapoptotic cell survival pathways: implications in chemo/ immunoresistance, rituximab unresponsiveness, prognostic and novel therapeutic interventions[END_REF], long half lives are less significant and possibly favoured as it allows an extended window of therapy with no significant increase in cross reactivity. Certainly, for antibodies neutralising systemic factors such as vascular endothelial cell growth factor, VEGF [START_REF] Panares | Bevacizumab in the management of solid tumors[END_REF] or tissue necrosis factor, TNFa [START_REF] Tracey | Tumor necrosis factor antagonist mechanisms of action: a comprehensive review[END_REF], a long half life is desirable.

Clearly there is no ''one size fits all'' form of conjugation when manipulating pharmacokinetics. A pharmacokinetic profile needs to be achieved that extends the longevity of the antibody, allow a more level range of bioavailability, enhances potency, and directs its particular mechanism of action specifically at the target site.

Chemical conjugation

Random (amine) versus site-specific (thiol) coupling

Chemical conjugation takes advantage of various reactive groups within the primary antibody sequence. Traditionally, lysine residues represent the favoured points of conjugation as they tend to have a high surface propensity. However, the presence of any given functional group does not automatically predispose it as a conjugation point. Reactive groups may be buried within the core tertiary structure of the antibody or sterically hindered and therefore unavailable for conjugation. Amino acids, essential for the antibody's function represent undesirable sites for conjugation. While some success has been observed through nonsite specific conjugation it has now become the standard to engineer proteins that eliminate residues where conjugation is undesired and re-engineer specific sites where conjugation is desired, particularly thiol-containing cysteines [START_REF] Kaushik | CDP-870 (certolizumab) in rheumatoid arthritis[END_REF][START_REF] Junutula | Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index[END_REF]). This has become increasingly favoured for conjugation purposes as, unlike the ubiquitous nature of amine groups, surface-available thiols tend to be present on proteins less frequently. In antibody Fab fragments, a free thiol is often found at the C-terminus of the light chain (a remnant from the whole immunoglobulin structure) or in other recombinant fragments, this can easily be introduced. Such engineering has three effects; it eliminates undesirable conjugates which would be detrimental to protein activity, secondly a more homogeneous (form of?) conjugates with specific conjugate isoforms are formed leading to more effective pharmaceutical characterisation, and thirdly, the number of reactive sites can be engineered to generate various conjugate to protein ratios with potentially different pharmacokinetics. Amine-directed chemical polysialylation (see below) was successfully used to modify an anti-tumour Fab [START_REF] Constantinou | Modulation of antibody pharmacokinetics by chemical polysialylation[END_REF]), but the same process damaged an anti-tumour scFv which was resolved by site-specific modification [START_REF] Constantinou | Site-specific polysialylation of an antitumor single-chain Fv fragment[END_REF]. Similarly, conjugation of larger polymer chains using amine-reactive conjugation of an anti-TAG-72 scFv was also found to be more detrimental on antibody activity than conjugation to carboxylic acid moieties using PEG-hydrazine chemistry [START_REF] Lee | Prolonged circulating lives of single-chain Fv proteins conjugated with polyethylene glycol: a comparison of conjugation chemistries and compounds[END_REF]. Unfortunately, in the absence of structural information it is difficult to predict which residues may be important for bioactivity. While some amino acid modifications can be tolerated, others, particularly within the complementarity-determining region (CDR), may be detrimental. If known, sensitive conjugation sites can be eliminated by mutagenesis. A non-essential lysine residue in the VH CDR3 of the C6.5 scFv was mutated to a non-conjugatable alanine residue to prevent unwanted chemical modification of HER2 binding site [START_REF] Adams | Delivery of the alpha-emitting radioisotope bismuth-213 to solid tumors via single-chain Fv and diabody molecules[END_REF].

Conjugates with modulating proteins and peptides

Chemically conjugating antibody fragments to other proteins which have a long serum half-life would, on the face of it, seem like an appropriate strategy. However, there is very little of this with most approaches focussing on recombinant fusions (see below). Due to its 19 day serum half-life (similar to immunoglobulin's 21 days, both interacting with the neonatal Fc-receptor) albumin is a common pharmacokinetic modulating protein partner. Albumin is a 67 kDa monomeric molecule and is the most abundant protein in blood serum (around 45 mg/ml in humans). Its low pI means that it has a net negative charge in the blood which hampers kidney filtration due to the anionic kidney basal membrane. The advantages of using albumin in this way outweigh the cross-reaction and side effects from Fc-mediated interactions. The most notable example of this is the work of [START_REF] Smith | Prolonged in vivo residence times of antibody fragments associated with albumin[END_REF] who evaluated three different albumin-binding strategies. Chemical conjugation of rat serum albumin (RSA) with an anti-TNFa Fab retained immunoreactivity but increased its bioavailability 17-200-fold. The same group also looked at a bispecific Fab which bound to endogenous RSA which produced an 8-fold increase in bioavailability.

Conjugates with modulating small molecules A 'portable', small albumin-binding molecule called 'Albu tag', which interacts with serum albumin and extends the half life of imaging agents, [START_REF] Dumelin | A portable albumin binder from a DNA-encoded chemical library[END_REF]) and antibodies [START_REF] Trussel | A new strategy for the extension of the serum half-life of antibody fragments[END_REF]) has been isolated from a DNA-encoded chemical library. This molecule, a 4-(p-iodophenyl)butyric acid derivative, was isolated from a DNA-encoded combinatorial chemical library. The portability of the 'Albu tag' overcomes the problems seen with other attempts with small molecules such as loss of albumin binding upon chemical conjugation. The nanomolar (K d ) affinity of the tag is retained upon attachment.

Conjugates with hydrophilic polymers

A wide range of polymers and attachment chemistries have been studied for their pharmacokinetic modulatory properties (Figs. 1,2). Polymers are used because the number, length and structural complexity of the polymer can be varied so as to produce conjugates with differing properties. Polydextrans (PD), polyvinylalcohols (PVA), poly(styrene-comaleic acid), polysialic acid (PSA), polyglycerols, polyals and polyethelene glycol (PEG) are some of the polymers that have been investigated for pharmacokinetic conjugation purposes. Their high hydrophilicity is due to their ability to interact with water molecules in solution. As such these hydrated polymers assume a much greater apparent molecular weight than that which can be applied to the polymer alone. This can be exploited pharmacologically when conjugated to therapeutic molecules to mask immunogenicity of native proteins, provide improved solubility and modulate pharmacokinetics. As the most advanced carrier system technology to date, the physicochemical properties of PEG have been the most extensively studied. Each of its ethylene oxide subunits can support up to three molecules of water [START_REF] Sasahara | Temperature dependence of volume changes on glycine-PEG and L-alanine-PEG in aqueous solution[END_REF] resulting in an increased hydrodynamic volume that provides an effective molecular weight 5-10-fold greater than that of proteins or other macromolecules of a similar size [START_REF] Harris | Effect of pegylation on pharmaceuticals[END_REF][START_REF] Fee | Prediction of the viscosity radius and the size exclusion chromatography behavior of PEGylated proteins[END_REF]. Furthermore, the length of polymer and its conformation as linear or branched dictates how the polymer behaves pharmacokinetically [START_REF] Yamaoka | Distribution and tissue uptake of poly(ethylene glycol) with different molecular weights after intravenous administration to mice[END_REF][START_REF] Yamaoka | Fate of water-soluble polymers administered via different routes[END_REF][START_REF] Fee | Size comparison between proteins PEGylated with branched and linear poly(ethylene glycol) molecules[END_REF][START_REF] Gursahani | Absorption of polyethylene glycol (PEG) polymers: the effect of PEG size on permeability[END_REF]. Generally, while clearance rates decrease as polymer-conjugate size increase, their permeability into potential target sites also slows [START_REF] Yang | Tailoring structure-function and pharmacokinetic properties of single-chain Fv proteins by sitespecific PEGylation[END_REF][START_REF] Cohen | Effect of chemical modification of recombinant human acetylcholinesterase by polyethylene glycol on its circulatory longevity[END_REF][START_REF] Gursahani | Absorption of polyethylene glycol (PEG) polymers: the effect of PEG size on permeability[END_REF]. Additionally, the charge associated with polymers and their effect on altering the charge on surface residues where conjugation takes place, can affect the overall isoelectric point of subsequent conjugates, which additionally affects its pharmacokinetic properties. As such, the pharmacokinetics of any given conjugate may be modulated according to a desired pharmacokinetic effect by altering PEG length and/or conjugation ratios, as well as charge.

A systematic study by [START_REF] Yang | Tailoring structure-function and pharmacokinetic properties of single-chain Fv proteins by sitespecific PEGylation[END_REF] used 5, 20 and 40 kDa maleimide-PEG conjugates to investigate the bioactivity and blood clearance rates of an anti-TNFa scFv site specifically conjugated at one or both of two available thiol sites. Although BIAcore binding data suggested some loss of bioactivity, cytotoxicity assays showed similar neutralization IC 50 values for all conjugate variants. Pharmacokinetically, the half-life of conjugates increased as their PEGylation content increased, demonstrating up to 100-fold prolongation of circulating half-lives [START_REF] Yang | Tailoring structure-function and pharmacokinetic properties of single-chain Fv proteins by sitespecific PEGylation[END_REF]. When comparing various conjugation ratios using 2, 5, 10, 12 and 20 kDa polymers linked to either primary amines or carboxylic acids, Lee et al. found that an increase in polymer length was found to be more effective for extending serum halflife than using a corresponding increase in total PEG by using smaller polymer lengths at a higher conjugation ratio [START_REF] Lee | Prolonged circulating lives of single-chain Fv proteins conjugated with polyethylene glycol: a comparison of conjugation chemistries and compounds[END_REF]). These studies agree Reactivity & Characteristics PEG Carbonyl-Imidazole [START_REF] Beauchamp | A new procedure for the synthesis of polyethylene glycolprotein adducts; effects on function, receptor recognition, and clearance of superoxide dismutase, lactoferrin, and alpha 2-macroglobulin[END_REF][START_REF] Veronese | Surface modification of proteins. Activation of monomethoxy-polyethylene glycols by phenylchloroformates and modification of ribonuclease and superoxide dismutase[END_REF] N-terminal α-amines and lysine ε-amine group acylation leading to formation of carbamate linkages.

Reactivity to other residues such as histidines and tyrosines may also be observed depending on the reaction conditions and the pKa effects of neighbouring residues.

PEG Benzotriazole carbonate and PEG Succinimidyl carbonate are noted for forming imidazolecarbonate linkages with histidine residues.

Polymer backbone ester of PEG succinimidyl carbonate can be readily hydrolyzed leading to possible immunogenicity towards the remaining tagged conjugate if used in vivo.

PEG p-nitrophenyl carbonate [START_REF] Veronese | Surface modification of proteins. Activation of monomethoxy-polyethylene glycols by phenylchloroformates and modification of ribonuclease and superoxide dismutase[END_REF] PEG Trichlorophenyl carbonate [START_REF] Veronese | Surface modification of proteins. Activation of monomethoxy-polyethylene glycols by phenylchloroformates and modification of ribonuclease and superoxide dismutase[END_REF] PEG Benzotriazole carbonate [START_REF] Dolence | Electrophilic polyethylene oxides for the modification of polysaccharides peptides (proteins) and surfaces[END_REF] PEG Succinimidyl carbonate (Zalipsky et al., 1992;[START_REF] Miron | A simplified method for the preparation of succinimidyl carbonate polyethylene glycol for coupling to proteins[END_REF] Reactive amination, selective for Nterminal α-amines and ε-amine groups found on lysine residues via a Shiff base.

Hydrate form of the PEG aldehyde is more stable and therefore the preferred agent to use.

Carbamate Bond

Carbamate Bond

Amine Bond Amine Bond Amine Bond

Cl O C O P E G O Cl Cl O C O P E G O N N O 2 N O C O PEG O N N N O C O P E G O N O O O C CH2CH2 C O O PEG O N N N Cl Cl O P E G HN C O PEG O R NH C CH2 CH2 C O O P E G O R N N N Cl HN O P E G R PEG NH R O P E G H2C NHCH 2 n R
well with non-antibody studies such as those of [START_REF] Clark | Long-acting growth hormones produced by conjugation with polyethylene glycol[END_REF].

The beneficial role of using branched PEG chains over linear ones has also been investigated, with investigators agreeing that branch chain conjugates may offer greater circulatory half-lives than linear chain counterparts [START_REF] Harris | Multiarmed, monofunctional, polymer for coupling to molecules and surfaces[END_REF]. To this end, the first FDA-approved anti-TNFa PEGylated Fab Maleimide can react under physiological conditions but is not stable under aqueous conditions. PEG Vinyl sulfone [START_REF] Morpurgo | Preparation of characterization of poly(ethylene glycol) vinyl sulfone[END_REF]

H 2 C C H S P E G O O SCH 2 CH 2 S P E G O O R
May also react with lysine residues PEG Iodoacetamide [START_REF] Kogan | The synthesis of substituted methoxypoly(ethylene glycol) derivatives suitable for selective protein modification[END_REF] Reaction must be perfomed under dark conditions to limit the generation of free iodine that may react with other residues PEG o-pyridyl didulfide [START_REF] Woghiren | Protected thiol-polyethylene glycol: a new activated polymer for reversible protein modification[END_REF] Forms stable disulfide bonds except under reducing conditions.

Amide Bond

C NH R O (CH2) O PEG n PEG CH 2 CH 2 H 2 N PEG CH2 CH2 HN CH2 Glycoprotein PEG CH2 C HN NH2 O N P E G O O ICH 2 CH 2 NH PEG O SCH2CNH PEG R O R S S mPEG N S S PEG
Fig. 2 conitnued antibody certolizumab pegol (Cimzia) is marketed as a branched PEG conjugate. Recently, it has been shown that there was no significant difference between the viscosity radii of branched and linear PEG-proteins having the same total molecular weight of PEG adducts, suggesting that any differences observed in circulatory half-lives cannot be explained by differences in hydrodynamic volume or glomerular filtration [START_REF] Fee | Size comparison between proteins PEGylated with branched and linear poly(ethylene glycol) molecules[END_REF]). However, although both linear and branched polymers confer greater stability and protection to a protein, it has been shown than when exposed to proteolytic cleavage, branched PEG conjugates demonstrated an even greater resilience to cleavage than their linear polymer conjugate counterparts. This observation could, in part, explain why branched conjugates have a seemingly longer half-life in vivo compared to those with linear PEG chains [START_REF] Monfardini | A branched monomethoxypoly (ethylene glycol) for protein modification[END_REF]. Since the earliest demonstration in 1977 [START_REF] Abuchowski | Effect of covalent attachment of polyethylene glycol on immunogenicity and circulating life of bovine liver catalase[END_REF], therapeutic protein PEGylation has expanded into a major biotechnology industry (Fraser-Moodie 2008). Antibodies are now emerging from this pipeline, for example, AMG-885 (Amgen/UCB) an anti-sclerostin PEGylated Fab for bone loss treatment and CDP-791 (Imclone/UCB) an anti-VEGFR2 PEGylated di-Fab for solid tumours.

PEGylation chemistries

Whilst the desire to modulate antibody pharmacokinetics is clear, the type of conjugation and the ability to produce them in a homogeneous and reproducible manner is still being refined. There are many methods of activating polymers for conjugation via certain functional groups within proteins (Fig. 2). These methods have evolved over time to minimise problems encountered with their predecessors and are thoroughly covered by [START_REF] Roberts | Chemistry for peptide and protein PEGylation[END_REF]. So-called 'first generation' PEGylation chemistry, which preferentially targets N-terminal amines and the epsilonamine group of lysines, react to give carbamate or amine linkages between the protein and the PEG chain. These chemistries are often limited to low molecular weight moieties and lead to unstable linkages, PEG impurities, and a lack of selectivity in modification; as such they have been used with limited success. The subsequent use of unifunctional methoxylated PEG (mPEG) has since been used to minimize the effect of cross linking between conjugates. 'Second generation' chemistries that also exploit amine conjugation have minimized considerably the difficulties described above. These include the use of PEG-aldehydes/ hydrate (Fig. 2). Although not completely selective, conjugation is observed preferentially at primary amines via formation of a Schiff's base, which is subsequently reduced to give a stable secondary amine linkage [START_REF] Kinstler | Characterization and stability of Nterminally PEGylated rhG-CSF[END_REF]. PEG-carboxylic acids have been extensively used being more stable and are favoured due to their reactivity at near physiological conditions [START_REF] Zalipsky | Facile synthesis of a-hydroxy-xcarboxymethylpolyethylene oxide[END_REF]. Increasingly, conjugation through surface thiol groups have become more desirable as they offer greater conjugation selectivity than that of lysine residues (Fig. 2). Where no free thiols are available, one or more free cysteine residues can be recombinantly engineered to facilitate site-specific conjugation [START_REF] Goodson | Site-directed pegylation of recombinant interleukin-2 at its glycosylation site[END_REF][START_REF] Natarajan | Characterization of site-specific ScFv PEGylation for tumor-targeting pharmaceuticals[END_REF][START_REF] Constantinou | Site-specific polysialylation of an antitumor single-chain Fv fragment[END_REF]. PEG derivatives such as PEG-maleimide, PEG-vinylsulphone and PEG-iodoacetamide have all been used in conjugations to form stable thioether linkages [START_REF] Goodson | Site-directed pegylation of recombinant interleukin-2 at its glycosylation site[END_REF][START_REF] Morpurgo | Preparation of characterization of poly(ethylene glycol) vinyl sulfone[END_REF]. Stable disulfide linkages have similarly been achieved using o-pyridyl disulfide-PEG, however such linkages are readily broken in reducing environments [START_REF] Woghiren | Protected thiol-polyethylene glycol: a new activated polymer for reversible protein modification[END_REF]. Cimzia, for the treatment of Crohn's disease, recently became the first protein antibody conjugated using thiol-specific, PEG chemistry to be approved for use by the FDA (Singh et al. 2003).

The use of slow-release or hydrolysable PEGylation has also been investigated. Here, the bioactivity, previously lost upon conjugation, is restored over time [START_REF] Greenwald | PEG drugs: an overview[END_REF][START_REF] Lee | Drug delivery systems employing 1,6-elimination: releasable poly(ethylene glycol) conjugates of proteins[END_REF][START_REF] Zalipsky | Thiolytically cleavable dithiobenzyl urethanelinked polymer-protein conjugates as macromolecular prodrugs: reversible PEGylation of proteins[END_REF][START_REF] Filpula | Releasable PEGylation of proteins with customized linkers[END_REF]. However, this technology has not been applied to antibodies as generally, antibody immuno-reactivity is preserved upon polymer conjugation.

Of course, the same types of chemistries described above are not unique to PEG conjugates, and similar advantages and disadvantages can be cited for any other polymer activated with the same chemistry. For example polysialic acid (PSA) conjugation has been investigated using amine and thiol chemistries with similar observations made. PSA is a naturally-occurring biopolymer (a-2,8, 2,9 linked sialic acid/N-acetyl neuraminic acid) found as colominic acid (in bacteria) or PSA (in mammalian cells). PSA was proposed by [START_REF] Gregoriadis | Polysialic acids: potential in drug delivery[END_REF] to have biophysical properties similar to that of PEG such that its hydrophilicity could be used to modulate the half-life of proteins. Initially demonstrated in 1993 with fluoroscein and in 1996 with an enzyme [START_REF] Fernandes | Synthesis, characterization and properties of sialylated catalase[END_REF][START_REF] Gregoriadis | Improving the therapeutic efficacy of peptides and proteins: a role for polysialic acids[END_REF], this approach was extended to antibody fragments. In the first instance, amine-based reductive amination using a PSA-aldehyde conjugation to H17E2 Fab, an antibody against the oncofoetal tumour antigen, placental alkaline phosphatase, was shown to produce conjugates that retain activity. Several coupling ratios were investigated, and all demonstrated increased blood residency in vivo compared to the parental Fab and approaching that of the whole immunoglobulin. Interestingly, the longest or highest PSA substitution ratio was not the most effective [START_REF] Constantinou | Modulation of antibody pharmacokinetics by chemical polysialylation[END_REF]. However, the same reductive amination chemistry was applied to MFE-23, a single-chain Fv fragment directed against another oncofoetal antigen, carcinoembryonic antigen. This resulted in immunoconjugates of reduced blood clearance and high bioavailability, but poor immuno-reactivity. This was resolved using the site-specific method with a thiol-directed maleimide activated PSA [START_REF] Constantinou | Site-specific polysialylation of an antitumor single-chain Fv fragment[END_REF]).

Recombinant approaches

Antibody fragment engineering

The proliferation of antibody fragments [START_REF] Holliger | Engineered antibody fragments and the rise of single domains[END_REF], and alternative binding frameworks [START_REF] Ewert | Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable frameworks and structure-based framework engineering[END_REF]) have led to many recombinant approaches to pharmacokinetic engineering. The selection and characterisation of scFvs, diabodies, nanobodies, DARPins and anticalins is normally followed by re-engineering into bigger molecules such as immunoglobulins, SIPs [START_REF] Borsi | Selective targeting of tumoral vasculature: comparison of different formats of an antibody (L19) to the ED-B domain of fibronectin[END_REF] or artificially multimerised proteins [START_REF] Pluckthun | New protein engineering approaches to multivalent and bispecific antibody fragments[END_REF]. These larger proteins have a longer blood halflife as of course, immunoglobulins have a natural retention and clearance mechanism through Fcdomain:neonatal Fc-receptor (FcRn, salvage receptor) binding via the reticulo-endothelial system [START_REF] Ghetie | Increasing the serum persistence of an IgG fragment by random mutagenesis[END_REF][START_REF] Anderson | Perspective-FcRn transports albumin: relevance to immunology and medicine[END_REF]). There are many strategies for increasing or decreasing the affinity between immunoglobulin Fc and FcRn to increase or decrease blood serum half-life. These are beyond the scope of this review but well described in numerous recent publications.

Albumin fusion strategies

Antibody-albumin fusions have been studied as a way to both slow down and increase blood clearance (Fig. 1). ScFv-HSA fusions using a variety of linker peptides increased the blood residence times approximately 12-fold [START_REF] Smith | Prolonged in vivo residence times of antibody fragments associated with albumin[END_REF]). These fusions expressed well in Pichia pastoris pointing to a strategy for low-cost expression. A radiolabeled anti-CEA scFv-HSA fusion protein ('immunobulin', expressed in mammalian NS0 cells) was designed by Yazaki et al. for tumor biodistribution and imaging studies. This scFv-HSA fusion showed a dramatic increase in tumor uptake, persistent high tumour:blood ratios, and limited normal tissue uptake in comparison with the scFv alone [START_REF] Yazaki | Biodistribution and tumor imaging of an anti-CEA single-chain antibody-albumin fusion protein[END_REF]). After 72 h, 27% injected dose/gram was reached with a tumour:blood ratio of almost 19:1. This is significantly better than that seen with whole immunoglobulins. A series of anti CD3 (T-cell)/anti-CEA (tumour cell) retargeting bispeciic antibodies were also fused to HSA in order to prolong their half-life for immunotherapy applications [START_REF] Muller | Improved pharmacokinetics of recombinant bispecific antibody molecules by fusion to human serum albumin[END_REF]). These rather complex molecules were successfully expressed in HEK293 cells. Mammalian cells have been shown by the Kontermann lab and others including ourselves (unpublished) to express bispecific scFvs while yields from E. coli were very poor [START_REF] Wright | Phage display of chelating recombinant antibody libraries[END_REF]. These constructs, bispecific scFv-, single-chain diabody-and tandem scFv-HSA fusion proteins (scFv 2 -HSA, scDb-HSA, taFv-HSA) were all stable and had increased in vivo bioavailability, as shown by a 6-8-fold increase in the blood exposure time. In vivo therapeutic benefit of such increased residence time is yet to be shown. Conversely, 'HSAbodies' are highly glycosylated scFv-albumin fusion proteins which have accelerated blood clearance making them appropriate for 2-step drug delivery systems [START_REF] Huhalov | Engineered single chain antibody fragments for radioimmunotherapy[END_REF].

Albumin-binding strategies

Rather than utilising the long residence time of albumin directly, there have been many recombinant approaches that ''piggy-back'' on albumin, in a similar way to the albu-tag described above.

Using peptide phage display, [START_REF] Dennis | Albumin binding as a general strategy for improving the pharmacokinetics of proteins[END_REF] identified a specific core sequence (DICLPRWGCLW) which bound to albumin with high affinity (K d 40 nM). They generated a peptide called SA21 that noncovalently bound to albumin with 1:1 stoichiometry at a site distinct from known small molecule binding sites (Fig. 1). Recombinantly fusing the peptide to an antitissue factor Fab (D3H44) significantly reduced the Fab in vivo clearance, and achieved 25-43% of the albumin half-life in mice and rabbits [START_REF] Dennis | Albumin binding as a general strategy for improving the pharmacokinetics of proteins[END_REF]. For tumour targeting, they incorporated the albumin-binding peptide to the Herceptin-derived Fab (Fab4D5), and showed a significant improvement in tumour deposition and retention, high tumor to blood ratios compared with Fab alone. This Genentech technology is known as AB.Fab, which has the advantage of cheap material expressed in E. coli. The authors also suggested that association with albumin leads to an altered route of clearance and metabolism [START_REF] Dennis | Imaging tumors with an albumin-binding Fab, a novel tumor-targeting agent[END_REF]. A possible refinement of this technology is the ability to modulate the pharmacokinetics of AB.Fabs by altering the affinity of the peptide, which was demonstrated using a range of peptides with K d values from of 4 nM to 2.5 mM [START_REF] Nguyen | The pharmacokinetics of an albumin-binding Fab (AB.Fab) can be modulated as a function of affinity for albumin[END_REF].

Recently, another group applied the same albumin binding strategy to a bivalent anti-epidermal growth factor receptor (EGFR) nanobody. They also illustrated improved tumor uptake (as high as cetuximab), and reduced blood clearance rate [START_REF] Tijink | Improved tumor targeting of anti-epidermal growth factor receptor nanobodies through albumin binding: taking advantage of modular nanobody technology[END_REF]). An alternative albumin-binding approach is to fuse a homologous albumin-binding domain (ABD3-46 amino acids/6 kDa) from streptococcal protein G with a recombinant antibody. ABD3, has a broad albumin species specificity, and interacts with HSA with K d of approx. 4 nM [START_REF] Johansson | Structure, specificity, and mode of interaction for bacterial albuminbinding modules[END_REF][START_REF] Linhult | Mutational analysis of the interaction between albumin-binding domain from streptococcal protein G and human serum albumin[END_REF]. [START_REF] Stork | A novel trifunctional antibody fusion protein with improved pharmacokinetic properties generated by fusing a bispecific single-chain diabody with an albumin-binding domain from streptococcal protein G[END_REF] applied this strategy to a bispecific single-chain diabody (scDb anti-CEA/anti-CD3) developed for retargeting of cytotoxic T cells to carcinoembryonic antigen (CEA) expressing tumor cells. They successfully showed all three parts of the chimeric protein were functional with 5-to 6-fold increase of prolonged circulation time. The drawback seemed to be a decreased immuno-stimulatory activity compared to the diabody alone (scDb). In a similar approach, a radiaolabeled anti-HER2 dimeric Affibody molecule (14 kDa) was also tested with ABD fusion for improved therapeutic efficacy. Good cellular retention, and reduced renal uptake in comparision with the non-fused dimer molecule were observed [START_REF] Tolmachev | Radionuclide therapy of HER2-positive microxenografts using a 177Lulabeled HER2-specific affibody molecule[END_REF].

Other fusion strategies

As described above, the use of inert, hydrophilic polymers represents the major strategy for pharmacokinetic engineering. Certain amino acid polymers bear this property and advantageously can also be attached to protein by genetic fusion. [START_REF] Schlapschy | Fusion of a recombinant antibody fragment with a homo-amino-acid polymer: effects on biophysical properties and prolonged plasma half-life[END_REF] investigated a glycine-rich homo-aminoacid polymer (HAP) that had an increased hydrodynamic radius. They used anti-HER2 Fab 4D5 as a model system and fused 100 and 200 residues of a repetitive sequence (Gly 4 Ser) to its light chain. They showed that the 200 residue 'HAPylated' Fab acquired a hydrodynamic volume more than double that of the Fab alone, moderate rise in half-life, but lower than the enhancement made by the ABD fusion (see above). Compared with more hydrophilic polymers such a PEG or PSA, the coiled structure of HAPs may hinder its development. However, this moderate effect could be beneficial for specialized applications, such as in vivo imaging. Other sequences and more extended and hydrophilic polymer chains are currently under investigation. Poly-(Pro-Ala-Ser) fusions have been developed as alternatives (PASylation) which acquire a more hydrophilic characteristic and much enhanced hydrodynamic radius, leading to better pharmacokinetic enhancement (A. Skerra, unpublished work).

Recombinant glycosylation domains

The HAP approach opens up the idea of using other recombinant approaches to obtain hydrophilic biopolymers which could have pharmacokinetic benefits. Presently, long chain carbohydrates seem the only option and have the advantage of being attached to proteins in a site-specific manner. Neural cell adhesion molecule (NCAM/CD56), can be heavily polysialylated by up to 200 sialic acid residues with a unique a2-8 linkage in mammals [START_REF] Georgopoulou | Overexpression of the alpha2,6 (N) sialyltransferase enzyme in human and rat neural cell lines is associated with increased expression of the polysialic acid epitope[END_REF]. We have been exploiting this to develop recombinantly polysialylatable antibodies [START_REF] Deonarain | Biological materials and uses therof[END_REF]Chen et al., unpublished work) which could have major improvements over chemical modification techniques.

Discussion and conclusions

The benefits of improving the longevity of therapeutic proteins are wide-ranging, from clinical benefits in terms of better dosing regimens and improved side effect profiles to commercial by extending product lifecycles and increasing their patent protection period. The emergence of fragments and alternative binding frameworks will see an increased requirement for half-life improving technologies. There are many approaches available and their features are compared in Table 1. PEGylation is clearly the current leader but it also lends itself as a stabilising agent and provides a stealth immune evasion technique. In a study of Crohn's disease patients who had become intolerant, or no longer responsive to the market-standard infliximab (anti-TNF alpha humanised immunoglobulin), clinical data presented by UCB (Union Chimique Belge) showed certolizumab pegol was able to provide an effective and rapid clinical response in more than 60% of the group.

PEGylation technologies dominate the pharmaceutical product pipeline, with many agents due to come to market. However, the technology has matured, and in the mid to long term, a decline in PEG use is expected with the emergence of improved biodegradable, stealth polymer technologies. The primary driver for this is the concern that PEG is not biodegradable, and with use for chronic conditions PEGylated peptides or by-products could accumulate in tissues and cause unforeseen toxic effects and/or generate an immune response [START_REF] Armstrong | Antibody against poly(ethylene glycol) adversely affects PEG-asparaginase therapy in acute lymphoblastic leukemia patients[END_REF]). Kidney vacuolization has been observed as a toxic side effect due to the inability of renal cells to degrade the PEG polymer [START_REF] Bendele | Short communication: renal tubular vacuolation in animals treated with polyethylene-glycol-conjugated proteins[END_REF]). This has already lead to the establishment of preemptive next generation polymers aiming to supercede PEG and is indicative of the fast pace at which the technology concept is evolving.

Lipoxen's PolyXen uses polysialic acid (PSA), a biodegradable and biocompatible human polymer, and with four non-antibody drug candidates presently in early clinical development it is currently tipped to overtake PEG in leading the stealth platform [START_REF] Fraser-Moodie | Business Insights: delivery mechanisms for large molecule drugs: successes and failures of leading technologies and key drivers for market success[END_REF]. This is supported by Baxter's integration of Lipoxen's PolyXen technology for possible use with its propriety proteins by entering into an exclusive worldwide development and license agreement to develop improved, longer-acting forms of blood-clotting factors [START_REF] Fraser-Moodie | Business Insights: delivery mechanisms for large molecule drugs: successes and failures of leading technologies and key drivers for market success[END_REF].

Whilst the choice of polymer used for conjugation is paramount in producing conjugates with minimal side effects for subsequent clinical use other parameters must be taken into account and will continue to vary according to the drug being used and its target. As well as the size of the polymer, branch complexity, its charge effect, and the site(s) of conjugation will all influence how bioactive the drug remains, how well it reaches its target and how stable it remains. However, from a pharmaceutical approach, the ease in which conjugates can be produced in a large, efficient and consistent manner is also important. To this end further development in recombinant conjugation methods may provide an appealing long term objective for producing viable antibody fragment conjugates for therapy.

Fig. 1

 1 Fig. 1 Schematic diagram illustrating the different pharmacokinetic enhancing strategies. Key: blue line connections represent recombinant fusions and grey line connections represent chemical linkages. Strategies 1-3 work by increasing hydrodynamic radius (chemical PEG/polysialylation, HAP fusions and PSA carriers) whereas 4-9 utilize binding to the neonatal Fc receptor, indirectly (via albumin, albumin binding chemicals, peptides or proteins) or directly

Fig. 2

 2 Fig.2Conjugation chemistries used for PEGylation and other modification strategies. A variety of chemistries have been used to covalently modify antibodies. Illustrated here are the activated groups and the resulting linkages formed. Although primarily used for protein PEGylation, other agents can be attached c

Table 1

 1 Advantages and disadvantages of various pharmacokinetic enhancing strategies

	Approach name	Modulation type	Advantage	Concerns
	PEGylation	Chemical	Hydrophilic water attraction	Non-biodegradable
			Improved protein solubility and stability	Toxic accumulation
			method for extending	Immunogenic
			half-life	Protein inactivation
				Downstream processing
				Costs and yields
				Homogeneity control
	Poly-sialylation	Chemical	Hydrophilic water attraction Improved	Protein inactivation
			protein solubility and stability	Downstream processing
			Biodegradable	Costs and yields
			Potentially non-immunogenic	Homogeneity control
			Promising clinical data
		Recombinant	Hydrophilic water attraction	Homogeneity control
			Improved protein solubility and stability	Less advanced technology
			Biodegradable
			Potentially non-immunogenic
			Easier production
			Less downstream processing
	Albumin conjugation	Chemical	Potentially non-immunogenic	Protein inactivation
	or binding		Biodegradable	Downstream processing
			Impressive pre-clinical data	Costs and yields
				Homogeneity control
				Less advanced technology
		Recombinant	Hydrophilic water attraction	Less advanced technology
			Improved protein solubility and stability
			Biodegradable
			Potentially non-immunogenic
			Easier production
			Less downstream processing
	HAPylation	Recombinant	Hydrophilic water attraction	Low hydrophilicity
			Improved protein solubility and stability	Long protein polymers
			Biodegradable
			Potentially non-immunogenic
			Easier production
			Less downstream processing

BiotechnolLett (2010) 32:609-622

Acknowledgements The authors acknowledge funding from Imperial College, Cancer Research UK (Ref: C18960) and European Union FP6 (Immuno-PDT Ref: LSHC-CT-2006-037489) which fund MPD, AC and CC respectively. We thank David Leak for critically reading the manuscript.


M.D Kathryn R Wagner
email: kwagner@jhmi.edu

Noah Lechtzin

Daniel P Judge

Current treatment of adult duchenne muscular dystrophy

Patients with Duchenne muscular dystrophy (DMD) are living longer into adulthood due to a variety of improvements in health care practices. This growing patient population presents new therapeutic challenges. In this article, we review the literature on current treatment of adult DMD as well as our own experience as a multidisciplinary team actively caring for 23 men ages 19 -38 years of age. Approximately one quarter of our adult DMD patients have remained on moderate dose corticosteroids. Daily stretching exercises are recommended, particularly of the distal upper extremities. Cardiomyopathy is anticipated, detected, and treated early with afterload reduction.

Oxygen saturation monitoring, noninvasive positive pressure ventilation and cough assist devices are routinely used. Other medical issues such as osteoporosis, gastrointestinal and urinary symptoms are addressed. Current and future therapies directed at prolonging the lifespan of those with DMD will result in further increases in this adult population with special needs and concerns. These needs are best addressed in a multidisciplinary clinic.

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Duchenne muscular dystrophy (DMD) has long been considered a uniformly, fatal childhood disease. While there were few survivors of DMD beyond teenage years in the 1960s and 1970s, lifespan has been gradually extended in the past few decades [START_REF] Eagle | Survival in Duchenne muscular dystrophy: improvements in life expectancy since 1967 and the impact of home nocturnal ventilation[END_REF]. With many DMD patients now living into their late 20s and 30s, it is now appropriate to anticipate that a current child with DMD will not only reach adulthood but may live well into his fourth decade.

Improvement in survival with current treatments, and potentially enhanced by future therapies described in this issue, necessitates that physicians are informed regarding appropriate care for this emerging population. Although there is as yet scant data on outcomes, the consensus is that this care is best delivered by a multidisciplinary team. This article will review the literature and our experience caring for adults with DMD in a multidisciplinary clinic.

Patients with DMD are living longer into adulthood from a variety of factors. These likely include routine flu and pneumococcal vaccination, aggressive physical therapy and early use of antibiotics. Nocturnal ventilation has improved oxygenation, decreased atelectasis and has had a measurable effect on reducing morbidity and mortality [START_REF] Eagle | Survival in Duchenne muscular dystrophy: improvements in life expectancy since 1967 and the impact of home nocturnal ventilation[END_REF]. Other changes in practice such as increased spinal surgery, use of afterload reduction for cardiomyopathy and earlier and prolonged use of corticosteroids are also likely contributors to increased survival, but there is currently insufficient data to evaluate their effects. Corticosteroid use has doubtlessly increased the number of years of ambulation. However, a correlation between walking and survival has not been demonstrated [START_REF] Parker | Analysis of an adult Duchenne muscular dystrophy population[END_REF]. Perhaps one of the greatest contributors to increased survival for those with DMD is coordinated, multidisciplinary care.

Due to increasing numbers and specialized needs of adults with DMD, we have Nineteen of these patients are Caucasian, 3 African-American and 1 Asian-American. A seamless transition is made from pediatric specialists to adult specialists by concurrent clinics in the same Muscular Dystrophy Association (MDA) center where adult specialists are introduced to patients prior to assuming their care and where records are shared electronically. Patients and families are seen by a neurologist, pulmonologist, cardiologist, physical and occupational therapists, care coordinator and where appropriate, social worker and genetics counselor. Twice yearly visits are encouraged to keep abreast of changes in health status and to decrease the number of hospitalizations.

Neurologic Care

The neurologist acts as orchestrator of services and becomes, in many instances, the primary medical doctor. Although all adults with DMD should have identified a pulmonologist and cardiologist familiar with their disease, the neurologist coordinates the care of these specialists. For example, as discussed below, history taken by the neurologist on number of chest infections and signs of nocturnal hypoventilation is important in making a collective decision regarding cough and ventilation assist devices. Similarly, shortness of breath, peripheral edema and orthopnea are important considerations for discussion of afterload reduction with the cardiologist.

Corticosteroids: Glucocorticoid corticosteroids improve muscle strength and function in children with DMD treated from six months to two years as shown in randomized controlled clinical trials [START_REF] Griggs | Prednisone in Duchenne dystrophy: A randomized, controlled trial defining the time course and dose response. Clinical Investigation of Duchenne Dystrophy Group[END_REF][START_REF] Mendell | Randomized, double-blind six-month trial of prednisone in Duchenne's muscular dystrophy[END_REF]. The most effective dose appears to be 0.75 mg/ kg/ day [START_REF] Manzur | Glucocorticoid corticosteroids for Duchenne muscular dystrophy (Review)[END_REF]. Whether to continue corticosteroids as an adult, and at what dose, is a decision that the neurologist makes with the patient and family. The decision is difficult due to known systemic risks of long-term steroid use and unknown, but potential, benefits to respiratory and cardiac muscle. The 2006

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Cochrane review of corticosteroid use in DMD could not evaluate long term benefits from currently published studies [START_REF] Manzur | Glucocorticoid corticosteroids for Duchenne muscular dystrophy (Review)[END_REF]. While the conventional practice several years ago was to discontinue steroids when the patient began to use the wheelchair full time, many patients are continuing to use steroids indefinitely with the hopes that it provides benefit to muscles of respiration. There is some data to support respiratory benefit as measured by forced vital capacity; however, these trials were also in children and short term [START_REF] Griggs | Prednisone in Duchenne dystrophy: A randomized, controlled trial defining the time course and dose response. Clinical Investigation of Duchenne Dystrophy Group[END_REF][START_REF] Mendell | Randomized, double-blind six-month trial of prednisone in Duchenne's muscular dystrophy[END_REF].

In our adult DMD clinic, we do not initiate patients on steroids who have chosen not to take steroids or have discontinued them due to side effects or uncertainty of benefit. patients for a 10 year follow up and found that prednisone dose was decreased to a mean dose of 0.36 mg/ kg/ day [START_REF] Pandya | Long term daily prednisone therapy delays decline in pulmonary function and improves survival in patients with Duchenne dystrophy[END_REF].

Exercise: Most adults with DMD have very limited motor abilities. However, some upper extremity muscles, especially finger flexors, may be well preserved and afford the patient the important abilities to control a motorized wheelchair joystick, play electronic games and use

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the computer keyboard. Physiotherapy should concentrate on stretching the upper extremity muscles to minimize contractures. A daily stretching routine should include elbow flexors, forearm pronators, wrist flexors, and long finger flexors [START_REF]Parent Project Muscular Dystrophy, Current Options for Care and Management[END_REF]. Stretching of the lower extremities, (hip flexors, knee flexors and foot plantar flexors) should also continue past the period of ambulation and into adulthood as this provides relief from symptoms of stiffness and improves pain. In addition to stretching, patients benefit from therapy in the pool with assistance and support of the head and neck, where the buoyancy provides a degree of freedom of movement.

Cardiac management

As supportive care for individuals with DMD leads to greater longevity, both recognition and treatment of cardiac manifestations are increasingly important. Prior to the development of noninvasive methods to assess cardiac function, the incidence of myocardial dysfunction among those with DMD was thought to be low, but cardiomyopathy was often seen on autopsy [START_REF] Heymsfield | Sequence of cardiac changes in Duchenne muscular dystrophy[END_REF].

Today, detection of asymptomatic left ventricular dysfunction and arrhythmia allows early and potentially life-saving treatment. In our practice, screening for anticipated cardiomyopathy is routine and co-incident with the diagnosis of DMD.

Incidence of dilated cardiomyopathy (DCM):

In addition to its role in skeletal muscle, dystrophin plays an essential role in stabilizing the cytoskeletal complex within cardiac myocytes.

Alterations in several components of this complex are associated with DCM (with or without associated skeletal myopathy), including delta-sarcoglycan, desmin, and metavinculin [START_REF] Tsubata | Mutations in the human {delta}-sarcoglycan gene in familial and sporadic dilated cardiomyopathy[END_REF][START_REF] Goldfarb | Missense mutations in desmin associated with familial cardiac and skeletal myopathy[END_REF][START_REF] Olson | Metavinculin mutations alter actin interaction in dilated cardiomyopathy[END_REF]. Despite consistent absence of dystrophin, however, the cardiac phenotype among DMD patients varies from no discernible left ventricular (LV) enlargement or dysfunction to early onset DCM with heart failure. The overall incidence of DCM in DMD has been estimated to be 25% by 6 years of age, 59% by 10 years of age [START_REF] Nigro | The incidence and evolution of cardiomyopathy in Duchenne muscular dystrophy[END_REF]. By adulthood, nearly all people with DMD have cardiac involvement [START_REF] Nigro | The incidence and evolution of cardiomyopathy in Duchenne muscular dystrophy[END_REF][START_REF] Cox | Dystrophies and heart disease[END_REF]. As cardiomyopathy is an age-dependent disorder to which all DMD patients are genetically predisposed, there is a high likelihood of cardiac dysfunction over

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an affected individual's lifetime. Frequently, significant cardiac abnormalities predate symptoms [START_REF] Heymsfield | Sequence of cardiac changes in Duchenne muscular dystrophy[END_REF][START_REF] Nigro | The incidence and evolution of cardiomyopathy in Duchenne muscular dystrophy[END_REF]. Accordingly, serial non-invasive assessment of cardiac dysfunction and early therapeutic intervention is strongly recommended.

Risk factors associated with the onset and severity of DCM are not clear. Severity of cardiomyopathy is not directly proportional to skeletal muscle strength, as has frequently been observed in Becker muscular dystrophy (BMD) patients who have nearly normal limb strength and receive cardiac transplantation for cardiomyopathy. Recently, there has been a suggestion that the deletion location may be linked to DCM. Jefferies and colleagues recently reported that in 47 individuals with DMD or BMD there was a higher incidence of DCM in those with a deletion occurring in exons encoding the N-terminal portion of dystrophin, and lower incidence of DCM in those with deletions occurring in exons encoding the C-terminus of dystrophin [START_REF] Jefferies | Genetic predictors and remodeling of dilated cardiomyopathy in muscular dystrophy[END_REF].

Data from additional subjects and follow-up of these initial individuals will be important to further evaluate this possibility, which could be an important tool in predicting cardiomyopathy.

Methods of detecting DCM:

Scoliosis, positive-pressure ventilation, and flexion contractures may all contribute to difficulties in obtaining reliable data from cardiac imaging, and each of these factors should be taken into account when considering the best method of determining cardiac size and function. Options include echocardiography, magnetic resonance imaging (MRI), and multiple gated acquisition scan (MUGA). Additional methods, such as serum B-natriuretic peptide (BNP) or noninvasive impedance cardiography, may be useful, but at this point, there is not sufficient data to support their routine use in this population instead of more traditional techniques. Electrocardiography (ECG) is useful for determination of cardiac arrhythmia, but lacks both sensitivity and specificity in assessment of structural cardiac disease.

A normal ECG does not provide adequate evidence of normal cardiac size or function, and an abnormal ECG provides no quantification of cardiac dysfunction (if present). In our practice, we perform ECGs when there is concern for arrhythmias such as atrial fibrillation, supraventricular tachycardia, or heart block (which is less common). We do not routinely perform surveillance

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ECGs for evidence of cardiac enlargement or dysfunction, as their yield is relatively low in comparison to echocardiography.

Echocardiography allows determination of the left ventricular (LV) size, wall thickness, valve function, as well as both LV systolic and diastolic function. While it may be technically limited in some patients with DMD, the use of intravenous echocardiographic contrast agents may improve its accuracy and feasibility. As opposed to some other methods of cardiac imaging, echocardiograms can be done while the patient remains in a wheelchair, which is a significant benefit to advanced patients with poor pulmonary function and hip contractures. While interpretation of the ejection fraction can vary among qualified readers, methods used to quantify cardiac performance may limit such variability [START_REF] Wong | Estimating left ventricular ejection fraction from two-dimensional echocardiograms: visual and computer-processed interpretations[END_REF]. In addition, factors such as intercurrent illness or respiratory insufficiency may be taken into account during interpretation of the study.

Cardiac MRI provides one of the most accurate assessments of LV size and function, and also adds the ability to image focal myocardial scarring. However, it is necessary to gate images such that data are not indiscriminately acquired throughout the cardiac cycle. Cine-MRI can be accomplished by compiling multiple images from evenly spaced phases of the cardiac cycle in a cinematic display. Limitations of cardiac MRI include the ease with which motion results in artifact, high cost, and technical factors related to acquisition of images in a narrow compartment for individuals with DMD.

MUGA scanning is performed by radiolabeling red blood cells, then measuring the activity of the radiotracer inside the cardiac chambers in systole and diastole through an external camera. It is one of the most accurate and reproducible methods of determining LV ejection fraction [START_REF] Burow | Analysis of left ventricular function from multiple gated acquisition cardiac blood pool imaging. Comparison to contrast angiography[END_REF]. However, this method is limited in its ability to provide information about LV wall thickness or cardiac valve performance. As with cardiac MRI, limitations related to patient immobility may make this technique less desirable for some individuals.

The consensus statement from the 107 th ENMC International Workshop recommends that DMD patients have an echocardiogram and ECG at diagnosis, every two years to age ten, and

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annually after age ten [START_REF] Bushby | 107th ENMC international workshop: the management of cardiac involvement in muscular dystrophy and myotonic dystrophy[END_REF]. In our adult DMD practice we use echocardiograms yearly until cardiomyopathy is identified, and subsequently if there is a change in clinical status, such as worsening symptoms or arrhythmia, or if the results of the repeat echocardiogram are likely to change management. This practice is in accordance with published guidelines for all forms of dilated cardiomyopathy [START_REF] Cheitlin | ACC/AHA/ASE 2003 guideline update for the clinical application of echocardiography: summary article: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines[END_REF]. Although echocardiography may be less sensitive than MRI for determination of cardiac size and function, we find that it has significant advantages of cost and convenience for DMD patients. Our hope is that additional research will determine the role of advanced cardiac imaging techniques in this population.

Cardiac arrhythmia: Both bradycardic and tachycardic arrhythmias can occur in DMD.

Sinus tachycardia is the most common cardiac arrhythmia associated with DMD. Possible mechanisms contributing to resting tachycardia seen among adults with DMD include profound deconditioning, adrenergic stimulation, or reactive increase in heart rate in response to poor cardiac function.

The risk of life-threatening ventricular arrhythmia is increased for patients with low ejection fraction, as has been noted for DCM of any cause [START_REF] Bardy | the Sudden Cardiac Death in Heart Failure Trial (SCD-HeFT) Investigators, Amiodarone or an Implantable Cardioverter-Defibrillator for Congestive Heart Failure[END_REF]. Attempts to identify electrocardiographic indications of increased risk have also been performed. Among 84 individuals with DMD age 18.6 + 4.8 years, Corrado and colleagues reported no additional prognostic value of standard or signal-averaged electrocardiography, or 24-hour Holter telemetry monitoring [START_REF] Corrado | Prognostic value of electrocardiograms, ventricular late potentials, ventricular arrhythmias, and left ventricular systolic dysfunction in patients with Duchenne muscular dystrophy[END_REF]. In contrast, QT dispersion is a marker of myocardial electrical instability, and when increased, is an indication of elevation in risk of ventricular arrhythmia [START_REF] Barr | QT dispersion and sudden unexpected death in chronic heart failure[END_REF]. Yotsukura and colleagues performed QT variability testing in 67 people with DMD, average age 20.5 + 4.7 years, and showed that increased QT dispersion is a marker of ventricular arrhythmia in this population [START_REF] Yotsukura | QT dispersion in patients with Duchenne-type progressive muscular dystrophy[END_REF].

Implantable cardio-defibrillators (ICDs) are becoming more widely used for patients at highest risk of fatal or life-threatening ventricular arrhythmias. However, receipt of an ICD remains a very personal decision for those at risk of life-threatening ventricular arrhythmia.

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While many people with DMD meet the current standard in the United States of qualifying for an ICD on the basis of an ejection fraction less than 35%, published guidelines discourage use of ICDs for people with untreatable terminal illness or life-expectancy less than 6 months despite ICD placement [START_REF] Gregoratos | Task Force Members, ACC/AHA/NASPE 2002 Guideline Update for Implantation of Cardiac Pacemakers and Antiarrhythmia Devices[END_REF]. Furthermore, use of standard medications described below can improve the ejection fraction and decrease the risk of ventricular arrhythmia.

Prior to consideration of an ICD, it is our practice to use medical therapy to both improve cardiac function and decrease risk of arrhythmia. If medications are not successful in improving the ejection fraction to greater than 35%, or if symptomatic ventricular arrhythmia (syncope or presyncope) occurs, then risks and benefits of ICD are considered and discussed in detail with the patient and family. This is a difficult topic for adolescents and young adults, though for those at greatest risk of life-threatening cardiac arrhythmia, it can be quite successful at prolonging life [START_REF] Gradaus | Potential benefit from implantable cardioverter-defibrillator therapy in children and young adolescents[END_REF][START_REF] Sears | Young at heart: understanding the unique psychosocial adjustment of young implantable cardioverter defibrillator recipients[END_REF]. The risks of psychological harm, inappropriate firing, and procedural complications of addition to standard therapy [START_REF]Effects of enalapril on mortality in severe congestive heart failure. Results of the Cooperative North Scandinavian Enalapril Survival Study (CONSENSUS). The CONSENSUS Trial Study Group[END_REF]. Similarly, in a cohort of less symptomatic patients with DCM, the SOLVD trial showed 16% reduction in mortality with enalapril treatment compared to placebo [START_REF]Effect of enalapril on survival in patients with reduced left ventricular ejection fractions and congestive heart failure. The SOLVD Investigators[END_REF]. The SAVE trial randomized patients with recent myocardial infarction and ejection fraction less than 40%, but notably without symptoms of heart failure, to receive either captopril or placebo [START_REF] Pfeffer | Effect of captopril on mortality and morbidity in patients with left ventricular dysfunction after myocardial infarction. Results of the survival and ventricular enlargement trial. The SAVE Investigators[END_REF]. During the follow-up period of 42 months, those treated with captopril had both improvement in survival and reduced morbidity and mortality due to major cardiovascular events.

As a class, ACEi are associated with side effects, including cough and angioedema.

Because of this, studies testing the comparative benefits of angiotensin II-type 1 receptor blockers (ARBs) in DCM have been performed. ARBs appear to have lower incidence of side effects and similar effects on reduction in mortality as compared to ACEi [START_REF] Pitt | Effect of losartan compared with captopril on mortality in patients with symptomatic heart failure: randomised trial--the Losartan Heart Failure Survival Study ELITE II[END_REF][START_REF] Pitt | Randomised trial of losartan versus captopril in patients over 65 with heart failure (Evaluation of Losartan in the Elderly Study, ELITE)[END_REF].

Duboc and colleagues recently reported their analysis of ACEi prior to onset of LV dysfunction in patients with DMD [START_REF] Duboc | Effect of perindopril on the onset and progression of left ventricular dysfunction in Duchenne muscular dystrophy[END_REF]. They enrolled 57 people age 9.5 to 13 years with DMD and normal cardiac function to receive an ACEi (perindopril) or placebo in a double-blinded fashion. After 3 years, all participants received open-label ACEi and were followed for 2 more years. After 3 years there was no difference in cardiac function in the 2 groups. However, after 5 years, there were 8 people with ejection fraction less than 45% in the placebo group compared to 1 person in the ACEi-treated group (p = 0.02). Based on these results, the study authors suggest starting all patients with DMD on ACEi as early as 9 years age. By the time an individual with DMD is seen in our adult DMD program, if not already started on ACEi or ARB by our pediatric collegues, it is our practice to do so even if cardiac function is normal or nearly normal.

Similar to ACEi and ARB, there are many studies supporting use of beta-adrenergic receptor blockers in people with symptomatic DCM regardless of the etiology [START_REF]The Cardiac Insufficiency Bisoprolol Study II (CIBIS-II): a randomised trial[END_REF][START_REF]Effect of metoprolol CR/XL in chronic heart failure: Metoprolol CR/XL Randomised Intervention Trial in Congestive Heart Failure (MERIT-HF)[END_REF][START_REF] Packer | Effect of carvedilol on the morbidity of patients with severe chronic[END_REF]36].

This class of medications provides not only anti-arrhythmic therapy, but also improves ejection fraction and adverse ventricular remodeling [START_REF] Jessup | Heart Failure[END_REF]. In our practice, use of beta-blockade is standard for those with symptomatic heart failure and is added after ACEi or ARB for individuals with persistent asymptomatic LV dysfunction (ejection fraction less than 40%).

For DMD patients with symptomatic heart failure, several additional medications are likely to provide symptomatic benefit and perhaps improvement in survival. In such cases, use of medications such as spironolactone, loop diuretics, and digoxin should be considered. The use of warfarin anticoagulation has been studied in several trials of dilated cardiomyopathy, and to date, has only been shown to be of benefit in the setting of concomitant atrial fibrillation, recent anterior transmural myocardial infarction, or upon demonstration of an intracardiac thrombus [START_REF] Cleland | The Warfarin/Aspirin Study in Heart failure (WASH): a randomized trial comparing antithrombotic strategies for patients with heart failure[END_REF][START_REF] Sirajuddin | Anticoagulation in patients with dilated cardiomyopathy and sinus rhythm: a critical literature review[END_REF]. We do not empirically treat our DMD patients with warfarin in the absence of a clear indication for its use other than low ejection fraction.

Emerging cardiac therapy: Yasuda and colleagues recently reported work performed in a murine model of DMD, the mdx mouse [START_REF] Yasuda | Dystrophic heart failure blocked by membrane sealant poloxamer[END_REF]. In dystrophin-deficient cardiomyocytes, they showed reduced compliance and increased susceptibility to stretch-mediated calcium overload.

Testing whether these abnormalities could be attributable to loss of membrane integrity, they treated dystrophin-deficient mice with poloxamer 188, a compound previously shown to insert into artificial lipid monolayers and repair damaged biological membranes [START_REF] Lee | Surfactant-Induced Sealing of Electropermeabilized Skeletal Muscle Membranes in vivo[END_REF]. Remarkably, functional deficits in dystrophin-deficient cardiomyocytes were repaired by intravenous infusion of poloxamer 188, suggesting that membrane stabilizing therapies may be an rational therapy for DMD based on molecular dissection of disease pathogenesis [START_REF] Yasuda | Dystrophic heart failure blocked by membrane sealant poloxamer[END_REF].

Future trials investigating emerging therapies for more common forms of dilated cardiomyopathy such as calcium sensitizers, passive cardiac restraint devices, and other neurohormonal modulators, may identify improved therapies for this condition. In addition, there is an urgent need for targeted clinical studies of both pathogenesis and therapy of cardiomyopathy in dystrophinopathy patients.

Pulmonary Management

The majority of individuals with DMD eventually die from complications of respiratory muscle weakness [START_REF] Finder | Respiratory care of the patient with Duchenne muscular dystrophy: ATS consensus statement[END_REF]. This includes progressive restrictive ventilatory defects, chronic

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13 hypoventilation, and pulmonary infections [START_REF] Benditt | Respiratory support of individuals with Duchenne muscular dystrophy: toward a standard of care[END_REF]. As most people with DMD begin to develop respiratory muscle weakness in the second decade of life, nearly all adults will have respiratory compromise. The following section will briefly review respiratory muscle involvement in DMD, the evaluation and treatment of neuromuscular respiratory complications.

The principal muscles for inspiration are the diaphragm and external intercostals. These are assisted by the accessory muscles of respiration, the sternocleidomastoid and scalenes [START_REF] De | Functional anatomy of the respiratory muscles[END_REF].

While expiration during quiet breathing is a passive act, forced expiration and coughing require use of the expiratory muscles including the internal intercostals and the abdominal wall muscles.

When inspiratory muscle weakness begins, patients are able to increase their respiratory frequency and maintain adequate ventilation. However, as weakness progresses patients hypoventilate which results in elevated arterial CO 2 and hypoxemia. Weakness of expiratory muscles results in decreased speech volume and more importantly, inability to cough forcefully enough to clear airway secretions. This places patients at increased risk of pneumonia.

Respiratory Evaluation: Patients with DMD are generally wheelchair bound before they develop significant respiratory muscle weakness. After age 10 to 14, patients gradually begin to lose respiratory muscle function. Estimates of the rate of loss vary from 0.04 L/year to 0.74L/year or 2% to 39% predicted per year. The median loss in vital capacity is estimated to be 8.0% predicted per year [START_REF] Tangsrud | Lung function measurements in young children with spinal muscle atrophy; a cross sectional survey on the effect of position and bracing[END_REF][START_REF] Phillips | Changes in spirometery over time as a prognostic marker in patients with Duchenne muscular dystrophy[END_REF]. Because the loss of respiratory function is very gradual and patients have severe arm and leg weakness prior to respiratory muscle weakness, most patients have little shortness of breath and few respiratory complaints. Therefore clinicians need to perform routine respiratory evaluations in order to detect progressive respiratory muscle weakness and impending respiratory complications. The American Thoracic Society's consensus statement on DMD recommends evaluation by a respiratory specialist twice a year after wheelchair confinement, fall in Vital Capacity below 80%, or age 12 [START_REF] Finder | Respiratory care of the patient with Duchenne muscular dystrophy: ATS consensus statement[END_REF]. In our practice, frequency of pulmonary follow-up is highly variable. Patients who have stable respiratory issues

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may only need to be seen annually, while other patients may need to be seen multiple times in a year.

Evaluation should include a thorough history including questions regarding dyspnea, orthopnea, cough, difficulty with secretions, swallowing problems and sleep characteristics.

Lying supine places the diaphragm at a mechanical disadvantage, and puts additional loads on the diaphragm from redistribution of blood into the pulmonary vasculature and shifting the abdominal contents against the diaphragm [START_REF] Lechtzin | Spirometry in the supine position improves the detection of diaphragmatic weakness in patients with amyotrophic lateral sclerosis[END_REF]. As a result, patients with DMD may note orthopnea or may complain of difficulty sleeping. Hypoventilation is often more pronounced at night and can result in nocturnal hypercapnia, elevation of the arterial partial pressure of carbon dioxide, which can cause morning headaches.

Objective measures of respiratory function are important for prognosis and timing respiratory interventions.

There is some debate regarding the optimal tests for individuals with DMD but spirometry is the most standardized, best studied single test to obtain. Spirometry measures the forced vital capacity (FVC) and forced expiratory volume in 1 sec (FEV 1 ). The forced vital capacity is the volume of air expired from a full inspiration to a full exhalation. Other useful testing includes maximal inspiratory and expiratory mouth pressures, cough peak flow rates, arterial blood gas measurement, end tidal CO 2 (the measurement of exhaled carbon dioxide, which should approximate arterial CO 2 ), oxygen saturation, and polysomnography. Cough peak flow rates can be measured with a standard peak flow meter attached to a tight sealing face mask [START_REF] Benditt | Respiratory support of individuals with Duchenne muscular dystrophy: toward a standard of care[END_REF]. Flow rates below 160L/min are felt to place individuals at high risk for mucous plugging and respiratory insufficiency. Cough peak flows fall during acute illness and a cough peak flow of 270L/min can fall below 160 during an acute infection. Therefore, many recommend initiating a series of pulmonary interventions (see below) when cough peak flow rates reach 270 L/min [START_REF] Bach | Prevention of pulmonary morbidity for patients with Duchenne muscular dystrophy[END_REF]. Alveolar hypoventilation is defined by an elevated arterial CO2. The best measure is obtained from arterial puncture but this is painful and can be technically difficult in adults with DMD due to limb contractures. Therefore capnography, measurement of exhaled carbon dioxide,

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15 may be an attractive alternative in these patients, though its use has not been thoroughly evaluated in this patient population. Pulse oximetry is a simple method to assess oxygenation. DMD patients are at risk for mucous plugging and pneumonia, both of which can cause abrupt declines in oxygen saturation. It has been recommended that individuals with DMD and cough peak flow ≤270 L/min be equipped with an oximeter at home to monitor their oxygen saturation regularly.

Hypoventilation, atelectasis, or mucous plugging can result in oxygen desaturation. Patients should be instructed to initiate assisted cough techniques and assisted ventilation if their oxygen saturation falls below 95% [START_REF] Bach | Prevention of pulmonary morbidity for patients with Duchenne muscular dystrophy[END_REF].

As noted above, hypoventilation is likely to occur while sleeping well before it occurs during wakefulness. This can occur simply from muscle weakness but may be complicated by central or obstructive sleep apnea. The recommendations for ordering sleep studies vary but the American Thoracic Society Consensus statement recommends yearly polysomnography [START_REF] Finder | Respiratory care of the patient with Duchenne muscular dystrophy: ATS consensus statement[END_REF].

However, overnight studies in a sleep laboratory can be challenging for DMD patients due to problems with positioning and mobility. In our practice, we consider polysomnography if there are symptoms of sleep disturbance that may be respiratory in origin and daytime values for CO Though randomized trials of NPPV in DMD are lacking, there are observational studies showing benefits. Noninvasive ventilation has been shown to improve hypercarbia, hypoxemia [START_REF] Mohr | Long-term follow-up of nocturnal ventilatory assistance in patients with respiratory failure due to Duchenne-type muscular dystrophy[END_REF][START_REF] Simonds | Impact of nasal ventilation on survival in hypercapnic Duchenne muscular dystrophy[END_REF], survival [START_REF] Eagle | Survival in Duchenne muscular dystrophy: improvements in life expectancy since 1967 and the impact of home nocturnal ventilation[END_REF], and quality of life [START_REF] Simonds | Impact of nasal ventilation on survival in hypercapnic Duchenne muscular dystrophy[END_REF], and may delay decline in lung function [START_REF] Vianello | Long-term nasal intermittent positive pressure ventilation in advanced Duchenne muscular dystrophy[END_REF].

Some advocates of noninvasive approaches to respiratory care in DMD, such as discussed above, argue that no DMD patients ever require tracheostomy [START_REF] Ishikawa | Nocturnal oxygenation and prognosis in Duchenne muscular dystrophy[END_REF][START_REF] Bach | Noninvasive ventilation is more than mask ventilation[END_REF] and that quality of life is better and complications are fewer when patients are managed noninvasively [START_REF] Bach | Noninvasive ventilation or paradigm paralysis?[END_REF].

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Nevertheless, this view is not universally accepted. Some patients find it easier to eat, drink, and speak with a tracheostomy than with various oral and nasal interfaces. Furthermore, there is concern that once a patient requires 24 hours ventilatory support, noninvasive ventilation is not as secure as a permanent tracheostomy. However, there are also disadvantages to tracheostomy. It requires surgery and the risks of anesthesia which are increased in this population. It requires hospitalization and is painful during the perioperative period and many patients have more pulmonary secretions shortly after having a tracheostomy tube placed. One of the more difficult factors to invasive mechanical ventilation is it requires that a caregiver be present 24 hours per day. Nursing support is not well covered by most insurance plans in the United States and this places both financial and emotional strains on families.

Decreased mobility and weakened cough place DMD patients at increased risk of complications from pulmonary infections. While there is no data regarding vaccination in this patient population, it is prudent to recommend pneumoccocal vaccination every five years and annual influenza vaccination.

Another important pulmonary intervention is assisted coughing. While manually assisted coughing can be of help, mechanical insufflation-exsufflation has been proven to produce greater cough velocities. Mechanical insufflation-exsufflation, is also known as cough-assist™, or an inexsufflator. It is a device that delivers a high positive pressure through a mask, mouthpiece, or tracheostomy, which inflates the lungs. Then, either manually or automatically it rapidly cycles to a negative pressure mode and sucks air from the lungs. This effectively simulates a natural cough and has been shown to generate high expiratory air flow. Studies have documented flows as high as 4.1 L/sec [START_REF] Gomez-Merino | Mechanical insufflation-exsufflation: pressure, volume and flow relationships and the adequacy of the manufacturer's guidelines[END_REF]. In order to prevent infections and mucous plugging, mechanical insufflation-exsufflation should be initiated when the cough peak flow falls below 270 L/min and should be used at least twice daily. Combining oxygen saturation monitoring, with NPPV and mechanical insufflation-exsufflation has been shown to reduce morbidity and mortality in DMD [START_REF] Bach | Prevention of pulmonary morbidity for patients with Duchenne muscular dystrophy[END_REF]. adolescence and there was no correlation between adult FVC in those that had received surgery (mean = 19% of predicted) and those that had not (mean = 22% of predicted). The emphasis in managing spinal deformity in adult patients is rather on proper seating. Proper wheelchair fitting and adjustments are critical to slow deformities, maintain mobility and enhance quality of life [START_REF]Parent Project Muscular Dystrophy, Current Options for Care and Management[END_REF][START_REF] Liu | Practical problems and management of Seating Through the Clinical Stages of Duchenne's Muscular Dystrophy[END_REF].

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Gastrointestinal and urinary concerns: Gastrointestinal symptoms may be under treated in this patient population. Nonambulatory DMD patients are more likely to suffer gastroesophageal reflux and esophagitis due to involvement of pharyngeal and hypopharyngeal musculature than healthy peers or ambulatory DMD patients [START_REF] Jaffe | Symptoms of upper gastrointestinal dysfunction in Duchenne muscular dystrophy: case-control study[END_REF]. Constipation due to lack of ambulation and smooth muscle hypomotility may decrease lung volumes and coughing force. It is important that the neurologist ask about these symptoms which in many cases, can be simply treated. Proton pump inhibitors are very effective in treating reflux with very rare side effects.

Good hydration, balanced dietary intake, docusate sodium (stool softener) and senna (a

Social Issues

grows. These issues are at least as important as the physical ones described above and include the need for quality care, personal independence, educational and employment opportunities, meaningful relationships and sexual activity. The ability to find and obtain practical aids is often difficult [START_REF] Parker | Analysis of an adult Duchenne muscular dystrophy population[END_REF] and can be aided by occupational therapists and care coordinators. A social worker is also an integral part of the multidisciplinary team. Social issue should be discussed with patients, solutions sought and potential side effects, such as depression, treated. Thirty percent of patients in our adult DMD clinic (7 of 23) are receiving treatment for depression with selective serotonin reuptake inhibitors (SSRIs). However, despite physical limitations, several studies demonstrate that adults with DMD perceive a high quality of life, one that is underestimated by health care professionals [START_REF] Bach | Life satisfaction of individuals with Duchenne muscular dystrophy using long-term mechanical ventilatory support[END_REF][START_REF] Rahbek | Adult life with Duchenne muscular dystrophy: Observations among an emerging and unforeseen patient population[END_REF][START_REF] Kohler | Quality of life, physical disability, and respiratory impairment in Duchenne muscular dystrophy[END_REF].

Conclusion

A variety of changes in multidisciplinary care have contributed to children with DMD surviving well into adulthood. This number of adults with DMD is likely to grow substantially from continued changes in practice such as early and prolonged use of steroids, nocturnal ventilation and ACE inhibitors. In addition, future therapies discussed in this issue including gene and cell based therapies, as well as efforts to increase muscle growth and regeneration, will not necessarily cure but attenuate the disease and further increase the number of DMD adults.

The collective clinical experience caring for this population is still small, and data sparse. It will be important to learn from these young men as we care for them.

  developed a multidisciplinary clinic dedicated to these patients. Patients were diagnosed by their pediatric neurologist by muscle biopsy showing lack of dystrophin immunostaining and/ or by A C C E P T E D M A N U S C R I P T genetic mutation analysis. Currently, there are 23 patients with a molecular diagnosis of DMD actively participating in our adult DMD clinic ranging in age from 18 to 38 (mean age 24).

  ICDs should be carefully considered in relation to the risk of sudden arrhythmic death for any patient considering an ICD. Of 23 adult DMD patients in our practice, one has received ICD and several are considering this therapy. Treatments for cardiac complications: In the absence of large clinical trials with adult DMD patients who have DCM, medical therapy is derived from multiple large clinical trials which enrolled varied groups of patients, including ischemic, valvular, genetic, and acquired forms of DCM. In these populations, clear benefits have been demonstrated with several therapies regardless of the etiology of DCM, making it a reasonable extension to use these medications in a population affected with DMD. Smaller clinical trials noted below have assessed treatment for patients with DMD more specifically. Use of angiotensin converting enzyme inhibitors (ACEi) is based on several large, well designed, prospective trials that show improved survival and decreased symptoms with this therapy for patients with DCM. The CONSENSUS-1 trial reported a 40% reduction in mortality at 6 months in patients with severe heart failure when treated with enalapril versus placebo, in A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 11

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 22 and FVC do not warrant ventilatory support If patients have daytime hypercapnia, oxygen desaturation or a sufficiently low forced vital capacity, we initiate non-invasive ventilation without a sleep study. Another crucial element of the pulmonary evaluation of individuals with DMD is discussion of advanced directives and attitudes regarding long term mechanical ventilation. Patients should understand the natural progression of DMD and should have ample opportunity to explore and learn about the various treatment options available. To this end, it can be helpful for patients to speak to other patients who have undergone tracheostomy and learn from their experiences. Respiratory Interventions/Treatments: Noninvasive positive pressure ventilators (NPPV) are devices that force air into the lungs via a mask or mouthpiece rather than an A C C E P T E D M A N U S C R I P T endotracheal tube or tracheostomy tube. The most common NPPV devices in use today are bilevel pressure cycled units in which separate inspiratory and expiratory pressures are set. The level of positive pressure support delivered to the patient is the difference between the inspiratory and expiratory pressure. Many patients with DMD have central sleep apnea and require NPPV machines that allow a respiratory rate to be set. As respiratory muscle weakness becomes more pronounced increasing levels of support are needed. Volume cycled ventilators, which are ventilators that deliver a preset tidal volume rather than a set pressure, may be able to provide more support than bilevel pressure cycled ventilators. Some practitioners favor these over bilevel pressure ventilators as respiratory muscle weakness progresses. Because nocturnal hypoventilation occurs before daytime hypoventilation, most patients initially use NPPV during the night and eventually shift toward daytime use as well. During daytime use, a mouthpiece can be a particularly convenient interface. Ventilators can be attached to wheelchairs and mouthpieces can be attached in place next to the patient's mouth. This allows patients to take breaths from the ventilator when needed but does not interfere with eating or speaking. The criteria to initiate ventilatory support are not well defined in DMD. One clear cut indication to initiate NPPV is evidence of hypercapnia or hypoxemia during sleep or wakefulness. Some advocate initiating NPPV when the cough peak flow falls below 270 L/min. While an optimal value of FVC indicating the need for NPPV has not been determined, most health care insurers in the United States reimburse NPPV units for restrictive ventilatory defects due to respiratory muscle weakness when the FVC falls below 50% predicted.

  promotility agent) are recommended to prevent severe constipation. On rare occasions, acute gastric dilation occurs with or without acidosis. This is a potentially life-threatening condition but, if recognized, responds well to prokinetic agents, nasogastric tube decompression of gastric contents and intravenous fluids.Urinary symptoms are common in both children and adults with DMD [62]. Many have urinary urgency and some have difficulty with daytime and/or nocturnal continence. Urinary symptoms should be evaluated by urodynamics, bladder ultrasound and urine microscopic analysis and culture as appropriate. Many will be aided by the use of anticholinergics such as oxybutynin[62]. Alternatively, a penile sheath attached to a collection bag may be used for urgency or incontinence and may be convenient for those who are away from home for any length of time.

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  In 23 adult DMD patients cared for in our multidisciplinary clinic, six (26%) of patients are unable to perform pulmonary function tests. In those who can perform spirometry, the mean vital capacity is 1.22 liters or 29% predicted. Ten (43%) of subjects currently use or have used noninvasive ventilation in the past and six (26%) of patients have tracheostomy tubes.Respiratory care for patients with DMD has improved dramatically over the last fifteen years. As a result both quality of life and survival have increased markedly. Patients with DMD need to have regular evaluations by pulmonologists, and need to be cared for by a multidisciplinary team that is fully familiar with noninvasive monitoring, ventilation, and assisted cough techniques. In spite of improvements in care, there are controversies regarding optimal methods for ventilation. These include debates over volume cycled ventilators vs. bilevel pressure ventilators, timing of ventilation and under what circumstances tracheostomy should be recommended. Further research will be crucial to resolve these clinical dilemmas.

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	Other Medical Issues Orthopedic concerns: Osteoporosis is a major problem in adults with DMD. This apparently occurs primarily from decreased weight bearing with loss of ambulation but may be accentuated by long term prednisone use. Fractures, particularly of the lower limbs, occur during routine daily care, including transfers, physical therapy and dressing and are estimated in one A C C E P T E D
	study to occur in as many as 30% of wheelchair-dependent DMD patients [56]. Wheelchair-
	dependent patients may have less exposure to sunlight and have low Vitamin D levels. For these
	reasons, we routinely treat all our adult patients, with daily calcium carbonate plus Vitamin D as
	well as weekly alendronate to decrease bone resorption.
	Scoliosis affects most patients with DMD and contributes to respiratory function
	compromise, pain and deformity. The decision to have surgical fixation of the spine is usually
	made in early adolescence and adults rarely have the orthopedic or cardiopulmonary criteria to
	consider surgery. Sixty percent of our adults DMD patients (14 of 23) had scoliosis surgery in
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Acknowledgements:

The authors wish to acknowledge Helen Posselt, Katie McGuire and the many young men and families with DMD for helpful discussions.

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