26 Drug Discovery World Spring 2014

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Acute Kidney Injury (AKI) is a complexmultifactorial disease with inflammatory,ischaemic, necrotic and apoptopic compo-nents simultaneously occurring, which rapidlycause damage and functional failure of the kidneys.Sepsis is the most common cause of AKI, account-ing for approximately 50% of cases, and sepsis-associated AKI (SA-AKI) is associated with short-and long-term risk of death5-7. During sepsis, theinitial host response to an infection becomes ampli-fied and then dysregulated, bringing the patientinto alternating hypo- and hyper-inflammatorystates. Several measures can be taken primarily topreserve haemodynamics in SA-AKI patients,including volume resuscitation and the use ofdiuretics, vasopressors and inotropes, which couldhave negative effects on the kidney. Once AKI

develops, there is no specific treatment and onlygeneral supportive measures are available. A newtherapy would thus be very welcome and should beaimed at targeting both inflammation and renalfunction impairment.

Renal replacement therapy (RRT)Currently, critically ill AKI patients are onlyoffered supportive care including the use of renalreplacement therapy (dialysis) to allow the kidneysto functionally recover. Supportive care is of limit-ed benefit because it does not improve or affectmarkers such as fluid balance, electrolytes andacid-base balance. Dialysis is also associated with anumber of problems such as the unwanted removalof minerals and vitamins as well as disrupting dos-ing and control of drug levels. Indeed, there is no

By Tim Knotnerus

Developing the firsttreatment for ACUTE KIDNEY INJURY

Acute Kidney Injury (AKI) is a devastating disease characterised by a rapid lossof kidney function resulting in inability to maintain fluid, electrolyte and acid-base balance. AKI is particularly common in patients admitted to the IntensiveCare Unit where incidence rates between 22% to as high as 67% of admissionshave been reported1,2. Even relatively small changes in serum creatinine arerelated to worse outcomes in AKI patients. Depending on the severity ofkidney injury, the mortality rate can be as high as 80%. Moreover, a third of allpatients who survive an episode of AKI develop chronic kidney disease, whichis accompanied by an enormous burden to both patient and healthcareproviders3,4. Despite an estimated 2 million AKI patients in the Western worldper year, there are no drugs approved for the treatment of AKI.

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Therapeutics

standard protocols or set of criteria to guide RRTintervention and the best time to start RRT is con-troversial because the only studies linking timingwith outcome are observational. Once RRT isstarted, uncertainty exists about when to stop8.Furthermore, no suitably powered randomisedcontrolled trials have been done to address thequestion on modality, ie intermittent or continuousforms of RRT. The appropriate intensity of RRT isalso uncertain, especially in critically ill patients, asindicated by two landmark studies conducted bythe Veterans Affairs/National Institutes of Health(VA/NIH) Acute Renal Failure Trial Network(2008)9 and The Renal Replacement TherapyStudy Investigators (2009)10. These concluded thatintensive renal support in critically ill patients withAKI did not decrease mortality, improve recoveryof kidney function, or reduce the rate of non-renalorgan failure, as compared to less intensive regi-men similar to usual-care practices.

Therapeutic strategiesDespite the significant unmet medical need andassociated commercial potential for new treat-ments of AKI, especially SA-AKI, no pharmacolog-ical treatment is currently approved and only a lim-ited number of candidate drugs or biological ther-apies are in clinical development. The multifactor-ial pathogenesis of the disease probably necessi-tates a multifactorial intervention to allow ade-quate response and durable restoration of kidney

function, which limits the number of potentiallyefficacious treatment options.

The only anti-inflammatory biological with sucha dual mechanism of action, which also showedsignificantly improved kidney function in twoPhase IIa trials, is Alkaline Phosphatase (AP). Thismembrane bound homodimeric enzyme exists inhumans as four different isoenzymes referred to asplacental, germ cell, intestinal, or tissue non-specif-ic AP. AP was originally considered as a novel ther-apy for sepsis because it dephosphorylates anddetoxifies endotoxin (lipopolysaccharide, LPS).

From the first Phase IIa trial, conducted in 36ICU patients with sepsis, it became clear that infu-sion of a purified bovine form of AP displayedclinical activity particularly in those patients whowere at risk to develop or had already been diag-nosed with AKI11. The treatments ability to inter-vene in AKI may be due to APs dual mechanismof action via dephosphorylation of both LPS andendogenous inflammatory triggers such as extra-cellular ATP and ADP12. Dephosphorylating ATPand ADP to Adenosine, by AP, inhibits formationof platelet-neutrophil aggregates, inhibits leuko-cyte endothelial adhesion, transmigration andinhibits inflammatory activation. Improvement ofcellular stress and damage markers, and apoptoticproteins, indicate that this local anti-inflammato-ry effect is due to better control of the damageresponse in peritubular and tubular cells. To con-firm the beneficial effects of AP, a second Phase IIa

Figure 1aThe most widely-usedbiomarker for AKI is serumcreatinine, a breakdownproduct from muscle tissuethat is normally removed bythe kidney. Serum creatininelevels are stable in healthyindividuals but a suddenincrease is observed in an AKIsetting. A 50-100% relativeincrease in serum creatinine isthe threshold that definesstage 1 AKI (or a 0,3mg/dl or26umol/L absolute increase ora decreased urine output). Afurther increase of 100-200%and then >200% in serumcreatinine correspond to AKIstages 2 and 3, respectively

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study was conducted, which focused specificallyon patients with sepsis and AKI. In this study, 36sepsis-associated AKI patients received eitherbovine AP or placebo intravenously for 48hours13. AP treatment resulted in improvement ofkidney function, as measured and demonstratedby a significantly faster recovery of creatinineclearance, and displayed a strong trend towardsreduction in requirement and duration of dialysis.In addition, AP-treated patients showed a signifi-cantly reduced length of ICU stay and demon-strated signs of improvement of other organs asexemplified by a trend towards reduced need formechanical ventilation.

During the course of the study, both renal dam-age biomarkers (urinary levels of KIM-1 and IL-18) and anti-inflammatory biomarkers (blood lev-els of CRP, LBP, and IL-6) declined significantlyfaster and were maintained at low levels in the APtreatment group. This strongly suggested that infu-sion of AP not only exerted local efficacy in failingkidneys but also has systemic anti-inflammatoryproperties. This clinical activity is corroborated bythe efficacy of AP in multiple preclinical animalmodels of kidney injury, whether induced byischaemia-reperfusion injury or by endotoxin. Thephysiological response to treatment with AP fol-lows a very clear pattern by serum biomarkers andhistopathology: an immediate local suppression ofinflammatory processes in the microcirculationand underlying tissue (apparent by reduced leuko-cyte activation and extravasation, and reduced

inflammatory cytokine production), accompaniedby suppression of acute damage to the peritubulartissues. Within 24 hours this resulted in sustainedrenal blood flow, perfusion, creatinine clearanceand GFR.

The Dutch company AM-Pharma is translatingthe use of AP to clinical application in AKI patientsand has produced a recombinant human form ofAP (recAP) with improved characteristics com-pared to purified bovine AP, including enhancedstability and half-life. recAP has recently been test-ed in a Phase I trial in healthy volunteers, showinga clean safety profile and AM Pharma will soonstart a large Phase II trial in sepsis-associated AKIpatients in the US and Europe.

Another therapeutic programme in clinical devel-opment for AKI involves allogenic mesenchymalstem cell therapy for the treatment of ischaemia-reperfusion injury-induced AKI. The stem cells arederived from pluripotent cells resident in the bonemarrow, processed and expanded in vitro. The pri-mary mode of action is thought to be via paracrineand endocrine mechanisms because engraftment ofthe stem cells after differentiation into target cells isabsent or rare and fusion of these mesenchymalstem cells with renal cells is not observed14.Efficacy of stem cell therapy will depend on theexpression of both trophic actions via secretion ofgrowth factors such as VEGF, HGF, IGF-1 andSDF-1, as well as anti-inflammatory actions viachemokine and cytokine production and increase ofadhesion molecule expression14. In order to avoid

Figure 1bPatients who have an AKI

insult lose kidney capacity asmeasured by glomerular

filtration rate (GFR). By thetime an increase of serum

creatinine can be measured,already 50% of the kidney

capacity has been lost,progressing to 82% of lost

kidney capacity at AKI stage 3.Serum creatinine levels in

patients who survive anepisode of AKI typically return

to normal. However, theymight have lost a significant

portion of their kidneys sparecapacity, which predisposes

them to increased risk ofsubsequent episodes of AKI,

chronic kidney disease (CKD)or end stage renal disease

(ESRD) in which patients areon permanent dialysis or

require kidney transplantation

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flooding of pulmonary circulation with large num-bers of mesenchymal stem cells, the administrationroute of the cells is through the distal thoracicaorta, accessed through the left carotid or femoralartery. For specific homing to the site of injury thecell therapy appears to rely on the expression ofCXCR4 and CD44, since their respective ligandsSDF-1 and hyaluronic acid are upregulated in AKI.Earlier attempts to use cell-based therapies for thetreatment of AKI have failed, mostly due to unex-pected side-effects. The US company Allocure iscurrently developing stem cell therapy for AKI,seemingly justified by the hypoimmunogenic natureof mesenchymal stem cells and its related use inallogenic protocols. From the companys Phase Istudy in 16 cardiac surgery patients who were atrisk of developing AKI, the stem cells appeared safeand well tolerated. Allocure noted a trend towardslower AKI incidence, reduced length of hospital

stay and reduced hospital readmission rates com-pared to a cohort of historical controls. The com-pany is currently conducting a Phase II trial in 200ischaemia-reperfusion injury-induced AKI patients.

Prevention strategiesFrom a trial design perspective, there are argumentsto developing a prophylactic approach for AKI dueto the known medical interventions (timed insult)that may lead to AKI. It is therefore possible todesign trials in which treatment can be started priorto such medical interventions. The relatively lowincidence rate, however, requires a substantial num-ber of patients to be included in the trial, to ensurea sufficient number of AKI events. This is a cleardownside of prevention trials and most preventionstudies appear to be underpowered.

Another novel experimental approach for theprevention of AKI after ischaemic reperfusion

References1 Hoste et al. RIFLE criteriafor acute kidney injury areassociated with hospitalmortality in critically illpatients; a cohort analysis.Crit. Care 10, R73 (2006).2 Thakar et al. Incidence andoutcomes of acute kidneyinjury in intensive care units: aVeterans Administration study.Crit. Care Med. 37, 2552-2558(2009).3 Oppert et al. Acute renalfailure in patients with severesepsis and septic shock asignificant independent riskfactor for mortality: resultsfrom the German PrevalenceStudy. Neprol Dial Transplant23, 904-909 (2008).4 Chawla et al. The severity ofacute kidney injury predictsprogression to chronic kidneydisease. Kidney Int 79, 1361-1369 (2011).5 Uchino et al. Acute renalfailure in critically ill patients: amultinational, multicenterstudy. JAMA 294, 813-818(2005).6 Ali et al. Incidence andoutcomes in acute kidneyinjury: a comprehensivepopulation-based study. J AmSoc. Nephrol. 18, 1292-1298(2007).7 Bagshaw et al. Septic acutekidney injury in critically illpatients: clinical characteristicsand outcomes. Clin. J. Am. Soc.Nephrol. 2, 431-439 (2007).8 Bellomo et al. Acute kidneyinjury. Lancet 380, 756-766(2012). 9 The VA/NIH Acute RenalFailure Trial Network. Intensityof Renal Support in CriticallyIll Patients with Acute KidneyInjury. N Engl J Med 359:1, 7-20 (2008). 10 The RENAL ReplacementTherapy Study Investigators.Intensity of Continous Renal-Replacement Therapy inCritically Ill Patients. N Engl JMed 361:17, 1627-1638(2009).11 Heemskerk et al. Alkalinephosphatase treatmentimproves renal function insevere sepsis or septic shockpatients. Crit. Care. Med. 37,417-423 (2009).

Continued on page 30

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Systemic and renal inflammation with impaired microcirculation in sepsis-associated acute kidney injury.

AP interacts with both the inflammatory and hypoxia pathway in AKI patients AP interacts with both the inflammatory and hypoxia pathway in AKI patientsSystemic Inflammation:Alkaline Phosphatase (AP) dephosphorylates lipopolysaccharide (LPS), thereby antagonising Toll-like receptor 4(TLR4) and preventing both systemic and renal inflammatory response. Extracellular adenosine triphosphate(ATP) may be converted by AP into tissue-protective and anti-inflammatory adenosine. Local Inflammation and Injury:Pathogens and inflammatory mediators (LPS and extracellular ATP) up regulate platelet-neutrophil aggregation,resulting in leukocyte transmigration into the interstitial compartment. This in turn causes swelling, coagulationactivation and vasoconstriction, which impairs microcirculation and causes hypoxia, all leading to kidney injury.

Figure adapted from Bonventre et al, Journal of Clinical Investigation 201118 and Peters et al, Am J Kidney Dis201419

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involves the use of selective bone morphogenicprotein (BMP) receptor agonists. BMP proteins areclosely related to the transforming growth factors(TGF), which are known to induce intracellularsignalling through Smad proteins. Expression ofthe BMP receptor activin-like kinase 3 (Alk3) iselevated early in diseased kidneys after injury15. Itsdeletion in the tubular epithelium leads toenhanced TGF-1-Smad signalling, epithelial dam-age and fibrosis, suggesting a protective role forAlk3-mediated signalling in the kidney. In mousemodels of kidney disease, BMP receptor agonistsselectively triggering Alk3 showed a potential ben-efit in controlling renal fibrosis. In contrast to theirpotential fibrotic effects, little is known about therole of BMPs on inflammation, which, as previ-ously discussed, is pivotal in the pathophysiologyof AKI. The Canadian company Thrasos is devel-oping small peptide BMP receptor agonists and hascompleted Phase I studies demonstrating safetyand useful pharmacokinetic profiles. A Phase IItrial is currently ongoing in patients scheduled forcardiac surgery considered at increased risk to

develop AKI. In this study, treatment will beadministered by four IV infusions, the first admin-istration prior to surgery.

An alternative option currently tested to preventAKI after cardiac surgery is the use of melanocyte-stimulating hormone (MSH), with potent anti-inflammatory activities. A modified alpha MSH-peptide analogue is in development targeting bothsystemic inflammation and apoptosis caused byhypoxia occurring during surgery. In vitro targetcharacterisation indicated that the peptide acts asan agonist on the melanocortin-receptor subtypes1, 3, 4 and 516. The modified peptide was origi-nally developed by Action Pharma, which hasbeen acquired by AbbVie. The peptide was testedin two Phase IIa studies, the first of which testedthe compound in 42 patients to assess potentialefficacy on cardiac surgery-induced systemicinflammation. Safety and tolerability were report-ed but no data on plasma concentration and expo-sure were published. In the second study, the can-didates were administered at two dose levels dur-ing surgery and early in the post-operative period.

Continued from page 29

12 Peters et al. Alkalinephosphatase as a treatment ofsepsis-associated acute kidneyinjury. J Pharmacol Exp Ther344, 27 (2013).13 Pickkers et al. Alkalinephosphatase for treatment ofsepsis-induced acute kidneyinjury; a prospectiverandomized double-blindplacebo controlled trial. Crit.Care. 16, R14 (2012).14 Togel et al. Kidneyprotection and regenerationfollowing acute kidney injury:progress through stem celltherapy. Am J Kidney Dis 60:6,1012-1022 (2012).15 Sugimoto et al. Activin-likekinase 3 is important forkidney regeneration andreversal of fibrosis. Nature18:3, 396-405 (2012).

Continued on page 31

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The trial focused on prevention of AKI and sys-temic inflammatory response in patients undergo-ing cardiac surgery on cardiopulmonary bypass.Although the primary endpoint, ie an absolutechange in serum creatinine levels compared tobaseline within the first seven days after surgery,or until discharge from hospital, was missed, thecompany reported positive data on a compositeendpoint of death, dialysis and kidney functionduring a 90-day follow-up. The compound is cur-rently being tested in a Phase IIb study.

Current guidelines for AKI essentially state thatthere is no evidence for the utility of diuretics inpreventing or treating AKI. Nevertheless, PLCMedical Systems RenalGuard, a medical devicewhich is designed to rapidly remove contrast dyesthat are known to be toxic to the kidneys, is beingsold in Europe. The companys position is that hav-ing high urine output does prevent contrastnephropathy provided that efforts are made to pre-vent dehydration. They do so by using a closed-loop fluid management system comprising a high-volume fluid pump and an intravenous urine col-lection system that interfaces with a standard Foleycatheter. An automated match performed in realtime reduces the side-effects associated with eitherunder-hydration or over-hydration. In an investiga-tor-sponsored study in Europe, RenalGuardreduced contrast-induced acute kidney injury from25% in the control group to 11% in theRenalGuard group17. RenalGuards pivotal studyis currently ongoing in the US. A number of otherstudies related to the use of diuretics is also underway, including a research study testing whetherprotocolised Diuretic Strategy (ProDiuS), a planfor adjusting diuretic doses based on measuredurine output, will improve clinical care for patientswith cardiorenal syndrome.

ConclusionAKI is a devastating disease with high incidence andmortality rates. Sepsis is the most common causeaccounting for approximately 50% of cases.Despite the enormous unmet medical need, to dateno pharmacological therapy has been licensed totreat or prevent AKI. As a consequence, only sup-portive care is currently being offered to these criti-cally ill patients, renal replacement therapy beingthe main option which allows the kidney time toheal but is also associated with a number of prob-lems. New therapies would thus be very welcome.Developing new treatments for this indication isparticularly challenging due to the multifactorialpathophysiology of the disease including inflamma-tory, ischaemic and direct nephrotoxic insults act-

ing simultaneously to rapidly cause functional fail-ure of the kidney. Fortunately, following successfulearlier studies, a number of large clinical trials withinteresting new approaches are under way. DDW

Tim Knotnerus joined the AM-Pharma team in2012 and has since been responsible for BusinessDevelopment of the company. Previously, Tim wasa senior associate at Aescap Venture which hejoined in 2008. He was instrumental in the financ-ing and support of various portfolio companiesincluding Avantium (the Netherlands), Biocartis(Switzerland), i-Optics (the Netherlands), EthicalOncology Sciences, now Clovis, (Italy) and to-BBB(the Netherlands). Tim gained both a Science andInnovation Masters and a Drug InnovationMasters with honors from Utrecht University.

Continued from page 3016 Doi et al. AP214, ananalogue of -melanocyte-stimulating hormone,ameliorates sepsis-inducedacute kidney injury andmortality. Kidney Int. 73, 1266-1274 (2008).17 Briguori et al. Renalinsufficiency following contrastmedia administration trial II(REMEDIAL II): RenalGuardsystem in high-risk patients forcontrast-induced acute kidneyinjury: rationale and design.EuroIntervention 6:9,1117-1122.18 Bonventre et al. Cellularpathophysiology of ischemicacute kidney injury. J Clin.Invest. 121:11, 4210-21 (2011).19 Peters et al. Alkalinephosphatase: a possibletreatment for sepsis-associatedacute kidney injury in critically illpatients. Am J Kidney Dis, (2014).

Alkaline Phosphatase and AM-Pharma historyAlkaline Phosphatase (AP) is an enzyme that is naturally present in many organisms,but is reduced in inflammatory indications such as Acute Kidney Injury (AKI) ,Inflammatory Bowel Diseases (IBD) and other conditions. In the early 1990sProfessor Poelstra and his group at Groningen University in the Netherlands dis-covered that one of the functions of this enzyme is to protect organs against inflam-mation and tissue damage via dephosphorylation of LPS and other potentially harm-ful substances. In 2001, AM-Pharma licensed an AP use patent from GroningenUniversity and started developing AP for inflammatory indications. AP isolated fromcalf intestines (bovine AP) was successfully tested in two phase II studies in sepsisand AKI patients and one Phase II study in moderate to severe Ulcerative Colitispatients. As a result of regulatory advice, conversations with potential partners andfuture supply logistics, the company decided to develop a fully human recombinantform of AP, not sourced from animals but produced using a controlled biotechno-logical manufacturing methods.

In 2011, AM-Pharma raised 29 million from an investor syndicate including theventure arms of AbbVie and Shire as well as six seasoned venture capital funds. Withthe newly-raised capital, AM-Pharma went on to develop a proprietary recombinantAP (recAP) which is highly stable and active, and has been optimised for treatinginflammatory conditions. It is being developed as an injectable for the treatment ofAKI and as an oral formulation for the treatment of Ulcerative Colitis. The enzymehas been produced by GMP manufacture for preclinical and clinical trial supply andcommercialisation. The company has recently completed a Phase I study in healthyvolunteers with varying doses of recAP showing a clean safety profile and favourablepharmacokinetics. A large, proof-of-concept and dose-finding, Phase III-enablingPhase II study in sepsis-associated AKI patients will start in 2014.