Lung Transplantation: Potential of Biomarkers & Nanotechnology in Management of Organ Rejection

Introduction

Lung transplantation is a therapeutic optionfor patients with end-stage chronic respiratory failure leading to dramatic improvements in both pulmonary function and health related quality of life and found to be an established therapy for selected patients with end-stage pulmonary disease.¹ Since the first successful lung transplant in 1983 by Dr. Joel Cooper and his team, over 42,000 recipients have benefitted from this procedure worldwide. Advances in surgical techniques, postoperative care and immunosuppression therapy have led to improved short- and long-term survival following lung transplantation.²

According to the registry of the International Society for Heart and Lung Transplantation (ISHLT) more than 55,000 adult patients received a LT in about 250 lung transplant centres from the early 90s. According to the report of this registry, adult patients who underwent primary LT between January 1990 and June 2014 had a median survival of 5.8 years, with unadjusted survival rates of 89% at 3 months, 80% at 1-year, 65% at 3 years, 54% at 5 years and 32% at 10 years and post-transplant survival has improved over time with a median survival of 4.2 years in the 1990–1998 era compared to 6.1 years in the 1999–2008 era.^3-5 

General indications for lung transplantation

The indications for lung transplantation can be broadly classified into the following main categories of end-stage lung diseases: obstructive lung disease, septic lung disease, fibrotic lung disease, and vascular lung disease. Of these categories, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), interstitial pulmonary fibrosis (IPF) and primary pulmonary arterial hypertension make up the most common indication in each category, respectively. Lung transplantation for pulmonary malignancy has also been shown to be effective in highly selected patients.^6-8

Types of transplant procedures:

• Single Lung Transplant (SLT)
• Bilateral Sequential Lung Transplant (BSLT)
• Living Lobar Transplant
• Heart-Lung Transplant (HLT)

Since the mid-1990s, the number of BSLT has consistently increased for all the major underlying disease categories. This is equally true for recipients with pulmonary fibrosis; rates rose from 20% in 1998 to 50% in 2010.⁹

When viewing the survival rates according to procedure type, BSLT appears to be better than SLT. These survival differences may be influenced by clinical factors such as age, underlying lung disorder, experience of lung transplant center, recipient co-morbidities and characteristics of procured donor allograft. The survival advantage may be more apparent in the later years of lung transplantation. The peri-operative mortality has been shown to be higher in the BSLT recipients; 1-month mortality 21% for BSLT versus 10% for SLT. Conditional half-lives (patients surviving at least 1year) are 9.4 years for BSLT versus 6.5 years for SLT. Short-term and long-term survival rates are also significantly related to recipient age. Patients less than 50 years of age have a 1-year survival of 80% compared to 72% in patients greater than 65 and a 5-year survival of 56% compared to 37% respectively.¹⁰

Post-operative complications after Lung transplantation:

Complications following lung transplantation may hinder allograft function and foreshadow patient survival. The five main complications after lung transplantation are primary graft dysfunction, post-surgical complications, alloimmune responses, infections and malignancy. Primary graft dysfunction, a transient ischemic/ reperfusion injury, appears as a pulmonary edema in almost every patient during the first three days post-surgery.Alloimmune responses represent acute rejection or chronic lung allograft dysfunction (CLAD). CLAD has three different forms namely bronchiolitis obliterans syndrome, restrictive allograft syndrome, acute fibrinoid organizing pneumonia. Infections are different depending on their time of occurrence. The first post-operative month is mostly associated with bacterial and fungal pathogens. From the second to sixth months, viral pneumonias and fungal and parasitic opportunistic infections are more frequent.¹⁰

Infection and primary graft dysfunction (PGD) are found to be the most common and devastating complications in the immediate postoperative period following lung transplantation (LT).11 According to the registry of the International Society for Heart and Lung Transplantation, in a total of 45,542 lung transplants the major reported causes of death within the first 30 days after transplantation were PGD (24.3%) and non-CMV infections (19.3%).12 Direct contact ofthe allograft with the environment, impaired clearance mechanisms caused by allograft denervation and profound immune suppression, especially in the first postoperative days, may be the reason for the high vulnerability of LT recipients to infection.

Infection found to be a common complication after lung transplantation, its recognition may be difficult, signs and symptoms may sometimes be misleading. Given that lifelong immunosuppression is obligatory to prevent acute and chronic rejection, immune system impairment contributes to increased patient vulnerability to infectious agents.^13,14 

Time found to be one of the factors affecting the category of infections the transplant patient can develop in the first post-operative month, the etiologic cause of the infection is often to be found in germs present in the donor or recipient. Nosocomial infections are found to be frequent in this period, as are infections related to technical problems [catheter infections, surgical site infections (SSI), dehiscence of bronchial anastomoses]. From 1 to 6 months after transplantation, opportunistic agents as well as reactivation of latent infections are common. Six months after transplantation, infections due to community-acquired pathogens are found to be the major concern.¹⁵

Early diagnosis of infectious complications after LT may allow prompt initiation of antimicrobial therapy and adjustment of immunosuppressant therapy which can help to prevent infectionrelated morbidity and mortality. These errors in the vulnerable patients may lead to harmful consequences thus demanding for the detection of rejection at earlier stages.¹⁶

Role of Biomarkers in early detection of lung rejection:

As a known fact Organ transplantation remains the single most effective treatment for end-stage organ failure and in 2014, approximately 30,000 organs were transplanted despite the well over 123,000 men, women and children needing the lifesaving operation. Consequently, detecting the onset of organ rejection is an important part of clinical management and is critical for the survival and health of the recipient. The invasive biopsy is the gold standard for diagnosing graft injury, yet it lacks sensitivity and specificity and detects pathological changes at advanced and irreversible stages of allograft damage. Thus there remains a need for noninvasive and predictive biomarkers that indicate damage when changes are occurring at the molecular level before changes in tissue morphology.

In medicine in general, a biomarker is a metric characteristic that reflects the severity or presence of some disease state. More generally, a biomarker is anything that can be used as an indicator of a particular disease state or some other physiological state of an organism. In 2001, the term biomarker was defined by a working group of the National Institutes of Health as “a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention.” Biomarkers include genes, proteins, genetic variations and differences in metabolic expression from different sources such as body fluids or tissues. They are characteristic biological properties that can be detected and measured in parts of the body such as blood or tissue.¹⁷

Solid organ transplantation is a lifesaving procedure performed to treat a multitude of health conditions. Unfortunately, transplant rejection an inflammatory response mediated by the recipient’s immune system remains a major and devastating challenge in the field. Because the individual transplant recipient possesses a very unique immunological landscape, much work has gone into making post-transplant care more personalized through the use of biomarkers that can herald rejection before it occurs or diagnose rejection more accurately than standard biopsy techniques.

Immunologic complications after lung transplantation (LT) include acute cellular rejection (ACR), antibody-mediated rejection (AMR) and most forms of chronic allograft dysfunction (CAD). ACR is an inflammatory process in which the reaction is mediated by the T-cell population. Most episodes of ACR fully recover with treatment, but repeated bouts are considered to be a risk factor for CAD. Biomarker cytokines interleukin IL-10, IL-15, IL-6, CCL5, CCR2 and IFNγ may play significant roles in this complication. CAD, the main limitation for longterm survival in LT, is characterized histologically by airway epithelial cell apoptosis and luminal fibrosis in the respiratory bronchioles causing airflow obstruction and, in some cases, lung parenchymal affectations causing restrictive lung disease. Several biomarkers have been studied in CAD, IL-6, IL-8, IL-17, IL-23, IL-13, IFN γ which may help in early detection of rejection of transplanted lung.¹⁸ 

miRNAs as biomarkers in transplant rejection:

The control of gene expression by microRNAs (miRNAs, miR) influences many cellular functions, including cellular differentiation, cell proliferation; cell development and functional regulation of the immune system.Invasive biopsies used in monitoring rejection are burdensome and risky to transplant patients. Novel and easily accessible biomarkers of acute rejection could make it possible to detect rejection earlier and make more fine-tuned calibration of immunosuppressive or new target treatment possible.

MicroRNAs (miRNAs, miRs) are a class of small (~22nt) noncoding molecules that inhibit translational initiation and stimulate decay of mRNA targets.

In lung organ rejection,biomarkers likemiR-369-5p, miR-144, miR-134, miR-10a, miR-142-5p, miR-195 and miR-155 helps in early detection of allograft rejection.^19-23

GeneralizedTreatment strategy after lung transplantation:

In large part, the success of solid organ transplantation lies in the appropriate utilization of immunosuppressive medications which act by suppressing the immune system there by reducing the risk of rejection of foreign bodies such as transplant organs. Immunosuppressive treatment of the transplantation patient begins with the induction phase, preoperatively and immediately after transplantation. Maintenance therapy then continues for the life of the allograft. Maintenance immunosuppression is the key for the prevention of acute and chronic rejections throughout the life of the graft.Immunosuppressive drugs can be classified as induction therapies, maintenance therapies and antirejection therapies. Maintenance immunosuppressive therapies include small molecule drugs (calcineurin inhibitors and antiproliferatives), fusion proteins and glucorticorticoids. Induction immunosuppressive agents consist of depleting and non-depleting protein drugs (polyclonal and monoclonal antibodies). Corticosteroids are the drugs used for induction and maintenance of immunosuppression, as well as for acute rejection. These agents prevent production of cytokines and vasoactive substances, including interleukin (IL)– 1, IL-2, IL-6, tumor necrosis factor-α, chemokines, prostaglandins, major histocompatibility class II, and proteases. The most common corticosteroids used in transplantation are oral prednisolone and intravenous methylprednisolone but suffers from many adverse effects like osteoporosis, avascular necrosis, cataracts, glucose intolerance, infections, hyperlipidemia, hypertension, etc.²⁴

Limitations of Current Immunosuppressive Agents:

Current lineup of immunosuppressive agents used are found to be associated with significant toxicity and side effects including nephrotoxicity, opportunistic infections, diminished tumorimmunosurveillance, serum sickness, anaphylaxis and neurotoxicity. Additionally, systemic delivery either oral (P.O.) or intravenous (IV) of these agents is often times associated with unpredictable blood levels of drug, leading to the presence of toxic (peak) or non-therapeutic (trough) concentrations.^32-34

Another barrier to improved patient outcomes after transplantation is the issue of patient compliance and non-adherence to the recommended treatment regimens.

Collectively, these shortcomings namely toxicity and non-adherence, highlight the importance of improving current therapies and developing future technologies to improve patient and graft outcomes in the milieu of transplantation. Such technologies would ideally be able to target delivery of therapeutic agents to the tissues/organs of interest (obviating systemic toxicity issues) and provide sustained release of factors (eliminating the need for regular and frequent consumption.^35-37

Role of nanocarriers for the efficient maintenance of immunosuppression:

The immune system is found to be a crucial defence mechanism in the body. Random non-specific systemic suppression of the immune system throughout the body can significantly increase the risk of infection. Nanoparticulate delivery of immunosuppressants can potentially elucidate specificity for immunosupressants to target cells and organs. Transplantation was introduced into medical practice in 1953 as a strategy for end stage organ disease.³⁸ However, graft rejection from mismatched donors limit the potential benefits of this strategy. This type of rejection is the result of a complex immune response to alloantigens expressed on the grafted cells.³⁹ In addition to T-cell activation, many reports indicate a role for B-cells in acute cellular and chronic humoral rejection.⁴⁰ Immunosuppressive agents have played an essential role in moderating the immune response to prevent the rejection and loss of the allograft and in increasing the survival of transplanted patients. However, severe side-effects are associated with chronic use of immunosuppressants. Infection by opportunistic organisms and primary pathogens, which also get a chance for growth due to general immunosuppression, are among the most common causes of death in transplant patients.However,immunosuppression found to be mainstay therapy in organ transplantation and autoimmune diseases besides its systemic immunosuppression and/or individual drug side effects. Hence, Nanotechnology approaches may be used to modify the mentioned shortcomings by enhancing the delivery of immunosuppressants to target cells of the immune system, thus reducing the required dose for function, and/or reducing drug distribution to non-target tissues.^41-45

The more commonly used immunosuppressants are calcineurin inhibitors (cyclosporine and tacrolimus) and their newer analogues (sirolimus and everolimus), inhibitors of nucleotide synthesis (e.g., azathioprine, mycophenolatemofetil, leflunomide), cytostatic agents affecting T-cell and B-cell division (e.g., methotrexate, mercaptopurine and gemcitabine), phosphodiesterase-4 (PDE4) inhibitors (mostly used as anti-inflammatory agents in COPD and autoimmune diseases), antibodies, and other biological approaches.⁴⁶

Calcineurin inhibitors Calcineurin inhibitors (CNIs) remain the cornerstone of immunosuppression after organ transplantation despite their adverse effects and CNIscurrently remain the mainstay maintenance immunosuppression of most clinically employed immunosuppression protocols. Due to toxic side effects associated with systemic CNI therapy and poor oral bioavailability, there has been significant interest in developing CNI based nanodelivery systems.^47-51

Cyclosporin (CsA) effectively suppresses immune reactions dependency on T cells, which are the key effector cells involved in graft rejection and autoimmunity. It forms a complex with cyclophilin, a cytoplasmic receptor protein present in T lymphocytes, and this complex then binds to calcineurin and inhibits Ca2+-stimulated dephosphorylation of the cytoplasmic component of the nuclear factor of activated T cells. This factor regulates transcription of numerous genes involved in T-cell activation and proliferation, such as IL-2, IL-4, and CD40 ligand.^52-54 When these genes are not expressed, T-cell-dependent immune responses are dramatically inhibited. However, CsA exhibits low oral bioavailability owing to its poor biopharmaceutical properties, such as its high molecular weight (1202Da), low solubility, low permeability, and high presystemic metabolism^{.55, 56} Consequently, there is an urgent need for the design and development of a novel formulation of CsA with better bioavailability and fewer side effects.⁵⁷

Nanoparticulate formulations of drugs can extend their blood circulation and offer new methods for targeted drug delivery after intravenous administration. Several nanoparticulateCsA formulations, such as emulsions, liposomes, microspheres, and polymeric nanoparticles (NPs), have been developed to compensate for the poor biopharmaceutical properties of CsAand potentially reduce its side effects through specific targeting and controlled release of CsA.^58,59 In particular, polymeric NPs based on polylactide (PLA) or poly(lactide-co-glycolide) (PLGA) have attracted much attention as delivery vehicles for CsA because of their excellent biocompatibility.^60-62

Cyclosporine nanoparticles prepared by encapsulating cyclosporine A (CsA) in poly(ethylene glycol)-b-poly(D,L-lactide-co-glycolide) (PEGPLGA) nanoparticles (NPs) by nanoprecipitation of CsA and PEG-PLGA found to have size less than <100nm in diameter with a narrow particle size distribution. CsA-encapsulated PEG-PLGA NPs were prepared in the solid form without noticeable change of properties by using Bovine serum Albumin. CsA released from the CsA/PEGPLGA-NPs suppressed the proliferation of T cells and the production of inflammatory cytokines in vitro.^{63, 64}

During the last decade, lipid nanoparticles (LN), which consist of a solid lipid matrix stabilized by surfactants, have gained considerable interest as suitable oral delivery systems for drugs that exhibit poor and variable gastrointestinal absorption, not only because of their adequate in vivo performance but also as a result of their versatility in manufacturing processes. Lipid nanoparticles enhance drug absorption, protectthe drug from possible biological fluid degradation, and allow controlled drug release and drug targeting.

CsA lipid nanoparticles prepared with low surfactantconcentration, likely to have low toxicity compared to commercial formulations. Developed formulations showed improved immunosuppressive effects in a stimulated human T-lymphocyte cell line when compared to conventional dosage forms.^65-69

Tacrolimus is a macrolide calcineurin inhibitor isolated from a strain of Streptomyces tsukubaensis. It is used as an effective immunosuppressant with a mechanism of action similar to that of cyclosporine.70-72 Multiple clinical trials have shown better efficacy and less severe systemic side effects for tacrolimus compared to cyclosporine.73-75Tacrolimus has been widely used in transplant patients, ocular immunologic disorders, and atopic dermatitis. Although, tacrolimus is approximately 99% protein bound, it widely distributes into most tissues including the lungs, spleen, heart, kidney, pancreas, brain, muscle, and liver .(76)Tacrolimus has a narrow therapeutic window and its half-life ranges from 11.7 to 34.8 h Moreover, it is a substrate of P-glycoprotein (P-gp) and cytochrome P450 3A4 (CYP3A4). Thus, any modulator of P-gp and CYP3A4 can change its PK.77-80Tacrolimus is poorly water soluble (4-12 μg/mL), its oral bioavailability is poor and shows high intra and inter-subject variability ranging from 4-93% (with a mean bioavailability of 17-22%.81, 82Low aqueous solubility, site dependent permeability, extensive first pass metabolism in the gut and liver, P-gp mediated drug efflux and influence of food are the most important reasons for low and variable oral bioavailability of tacrolimus.83-87

Nanoparticles may be used to correct some of these shortcomings and as a result enhance tacrolimus bioavailability (Table 3). While tacrolimus is available as asolution for injection, Prograf, the intravenous administration of tacrolimus is usually limited to early stages of organ transplantation and to cases where oral administration is not feasible.88,89

In graft rejection, antigen presentation occurs in the graft and lymphatics. Therefore, by targeting tacrolimus to the liver and spleen, graft survival could be achieved with a decrease in nephrotoxicity. Poly (lactide) tacrolimus nanoparticles (PLA-TAC-NP)formulated were tested in rats for targeting efficiency of tacrolimus to the liver and spleen. It has been found PLA-TAC-NP increased tacrolimus concentration in the liver 24 fold and in the spleen 1.94 fold whereas tacrolimus concentration in the kidneys was decreased by 7.12 fold. Tacrolimus in the form of nanoparticles can be promising drug delivery systems for achieving localized immunosuppression thereby minimizing nephrotoxicity in organ transplant patients which is one of the major side effect of tacrolimus when administered in conventional dosage form.⁹³

Sustained delivery of tacroimus from PLGAbased micro-nanoparticles is reported to be effective at preventing liver and islet allograft rejection and mitigating the effects of colitis in rodent models.

With the advent of biocompatible and biodegradable polymers, much research has focused on the development of suitable polymeric drug delivery systems and their design for sustained drug release applications. Polymeric drug delivery systems have the potential to protect drugs from degradation, and at the same time, provide their release at the targeted site in a predesigned manner to achieve more effective therapies while eliminating the potential for both under- and over-dosing. Polymeric drug delivery systems such as biodegradable polymer microspheres are simple to fabricate. Moreover, they offer facile administration via routes including oral, pulmonary and parenteral injection, and they do not need surgical removal upon complete drug release.⁹⁴

Tacrolimus Poly-D, L-lactic acid (PDLLA) releasing microparticles that (when administered orally) found to prolong small bowel transplant survival in a porcine model when compared to conventional dosage forms of tacrolimus. Significant research has also been conducted in the development of pH sensitive tacrolimus micronanoparticles which can deliver tacrolimus to the colon.⁹⁵

Innovations in nanotechnology and regenerative medicine have enabled the development of a hydrogel system that can be embedded in transplanted grafts which could facilitate sitespecific graft immunosuppression for long term graft survival while minimizing systemic immunosuppression and reducing the number of systemic drugs required. Triglycerolmonostearate (TGMS) enzyme-responsive hydrogel loaded with tacrolimusinjected subcutaneously or intramuscularly prevented hindlimb allograft transplant rejection more efficiently than conventional dosage form in a rat model.96 Prepared tacrolimus-loaded hydrogel found to have improved safety, efficacy, and patient compliance.

Conclusion

Lung transplantation can improve quality of life and prolong survival for individuals with endstage lung disease, and many advances in the areas of both basic science and clinical research aspects of lung transplantation have emerged over the past few decades. However, many challenges must yet be overcome to increase post-transplant survival, which include primary graft dysfunction, forms of cellular and antibody-mediated rejection, chronic lung allograft dysfunction, and infections.

Rate of graft loss due to acute rejection can be decreased by adequate immunosuppressive therapy, but however achieving adequate immunosuppression, even with today’s more effective drugs, remains a challenge that cannot be met for every patient.

Despite decades of advances in transplant immunology, tissue damage caused by acute allograft rejection remains the primary cause of morbidity and mortality in the transplant recipient. Moreover, the long-term sequelae of lifelong immunosuppression leave patients at risk for developing a host of other deleterious conditions. Controlled drug delivery using nanoparticles can be an effective way to deliver higher local doses of a given drug to specific tissues and cells while mitigating systemic effects.⁹⁷

Nanotechnology has shown great potential in improving the ADME properties as well as the therapeutic index of several major existing immunosuppressive agents in preclinical models. This is largely due to the large capacity of nano-delivery systems in the solubilization of poorly soluble immunosuppressant drugs, their ability for sustaining the rate of drug release upon systemic or local administration, and/or capability of nano-carriers in redirecting the encapsulated drug from normal tissues to sites of drug action in the RES system or inflamed tissues. As per the reports published, clinical studies on these systems are still at early stages and need more time to establish themselves.