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International Journal of Antimicrobial Agents 28 (2006) 90–94

Review

Recombinant human activated protein C

Martin Hughes ∗

Intensive Care Medicine, Royal Infirmary, Glasgow G2 4AQ, UK

bstract

This review will summarise the relevant pathophysiology of sepsis, the rationale for treatment with recombinant human activated proteinand the evidence for and against its use, and will provide evidence-based recommendations for its administration.2006 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

eywords: Drotrecogin alfa activated; Recombinant human activated protein C; Sepsis syndrome; Pathophysiology; Drug therapy

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. Introduction

Severe sepsis is increasingly recognised as an impor-ant cause of morbidity and mortality. In the USA, severeepsis causes ca. 215 000 deaths per year, which is asany as acute myocardial infarction [1]. The mortality rate

anges from 20% to 50% despite advances in intensive care2].

The first trial to report a reduction in mortality from sep-is was published in 2001 when the PROWESS study [3]f recombinant human activated protein C (drotrecogin alfactivated) demonstrated a 19% relative risk reduction. Sincehen there have been disagreements in the scientific commu-ity regarding the efficacy and safety of this very expensiveherapy [4,5]. This review will summarise the rationale forreatment and the evidence for and against its use and willrovide evidence-based recommendations for its administra-ion.

. Pathophysiology of sepsis

The response to infection begins with the host cellecognising components of microbial cell walls. These

omponents include lipopolysaccharide (endotoxin) fromram-negative bacteria, peptidoglycan or lipoteichoic acid

rom Gram-positive bacteria, and cell wall structures such

∗ Tel.: +44 141 211 4225.E-mail address: martinhughes@aol.com

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s flagellin or curli. Some Gram-positive bacteria alsoroduce potent exotoxins that induce massive T-cell acti-ation.

These cell wall structures are recognised by Toll-likeeceptors (TLRs). TLRs have a wide range of ligand speci-city including bacterial, viral and yeast proteins. For exam-le, endotoxin is recognised by TLR4, and Gram-positiveell wall structures are recognised by TLR2. Activation ofLRs triggers a complex host response that is still imper-

ectly understood.Initially there is a pro-inflammatory response where

ytokines (such as interleukin (IL)-1, tumour necrosis factor-lpha (TNF-�) and IL-6) induce activation of leukocytes,ndothelium and adhesion molecules. There is produc-ion of nitric oxide synthase, prostaglandins and acutehase proteins. Lipid mediators (such as platelet activat-ng factor, leukotrienes, thromboxane and tissue factor)ctivate the coagulation processes and the endothelium,hilst chemokines mobilise and activate neutrophils andacrophages. There is a reduction in physiological antico-

gulants such as protein C, heparin and anti-thrombin. Thexcess nitric oxide has several effects (Table 1) and some ofhese account for the clinical picture of a vasodilated patientho is unable to utilise oxygen.As well as stimulation of inflammation and coagulation,

here is inhibition of the normal regulatory fibrinolysis by

lasminogen activator inhibitor, so that clots are formed butot broken down.

This triad (coagulation, inflammation and reduced fibri-olysis) results in microvascular thrombosis. The subsequent

of Chemotherapy. All rights reserved.

M. Hughes / International Journal of Antim

Table 1Effects of nitric oxide

Vascular hyporeactivity and dilatationInhibition of mitochondrial respirationCytotoxic effectsReduced leukocyte adhesionIA

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ifTable 3. In 164 centres in 11 countries, 1690 patients withsevere sepsis of less than 24 h duration were randomised in adouble-blind fashion to receive drotrecogin alfa or placebo.This was a pragmatic trial and other aspects of ICU care

Table 2Effects of protein C

Antithrombotic Irreversible inactivation of factors V and VIIIAnti-inflammatory Reduces formation of TNF, IL-8, IL-6 and

thrombin; limits the rolling of monocytes andneutrophils on injured endothelium by bindingselectins; may reduce apoptosis

nhibition of platelet aggregationngiogenesis, proliferation of smooth muscle cells

eduction in blood flow may contribute to end-organ hypox-emia and damage.

Emphasis on the pro-inflammatory pathophysiology ofepsis led to an extensive search for anti-inflammatory agents.lthough agents were developed that were theoretically

ttractive and were successful in animal models, an overalleduction in human mortality in randomised controlled trialsroved elusive.

Some of the disappointing results for anti-inflammatorygents may have been related to the logistical difficulty ofdentifying and recruiting patients at an early enough stagef their disease. For example, anti-TNF or anti-IL-1 may beneffective once the detectable levels of TNF and IL-1 haveisappeared (ca. 90 min after the initial insult).

Other explanations for this failure included the conceptf ‘redundancy’: there are so many inflammatory path-ays that blockade of one of them may make that pathway

uperfluous—the many other inflammatory pathways wouldimply replace its function. Hence, it was not the theoryhat was thought to be wrong; it was the lack of an all-ncompassing anti-inflammatory agent.

In practice, although patients do die of an overwhelm-ng inflammatory response, it is not the usual mode ofeath. The most frequent mechanism of death in Intensiveare Units (ICUs) is withdrawal of therapy in a patientith multiple organ failure unresponsive to treatment [6].hese patients have features consistent with immune com-romise such as a predisposition to nosocomial infection,oss of delayed hypersensitivity and an inability to clearnfection.

. Immune failure, anergy and apoptosis

Following an initial pro-inflammatory burst, there is awitch towards an anti-inflammatory state [7]. For example,ndotoxin-stimulated whole blood from patients with sep-is releases less TNF-� and IL-1 than blood from controls8]. In addition to anti-inflammatory cytokine productionsuch as IL-10 and IL-4), there is evidence of anergic T-ells in sepsis. In patients with peritonitis, there is decreasedro-inflammatory T-helper cell activity but no change in anti-nflammatory T-helper cell activity, consistent with anergy

9].

Whilst cell necrosis generally results in inflammation, pro-rammed cell death (apoptosis) produces anergy. It has beenemonstrated that there is a profound progressive apoptosis-

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nduced loss of B-cells, CD4 T-cells and follicular dendriticells in patients who have died of sepsis [10–12].

Autopsy studies of patients who die in the ICU reveal a sur-rising discrepancy between the degree of organ dysfunctionnd cell death [10]. Despite profound multiple organ dys-unction, cell death in the heart, lung, liver and kidney waselatively minor. This is consistent with the observation thatost organ function returns to baseline if the initial insult

s survived. It has been suggested that much of the organysfunction in sepsis might be explained by ‘cell stunning’10,13]. It may be that sepsis activates defence mechanismshat cause cellular processes to be reduced to basic ‘house-eeping’ roles.

Therefore, there is both an inflammatory and anti-nflammatory/anergic reaction to sepsis. The precise nature ofhe response encountered in an individual patient may dependn the complex interplay of factors including host geneticesponse, the infecting organism, the site of infection andhe patient’s co-morbidity. The clinical picture ranges from auge inflammatory response in a previously healthy individ-al (for example meningococcal sepsis in a young person) to arumbling low-level illness with gradual onset of progressivergan dysfunction (for example a nosocomial chest infectionn an elderly patient with renal failure and diabetes). In future,t may be possible to characterise more accurately the state ofhe response to an infection (the ‘cytokine screen’) as well ashe predisposition to certain responses (the ‘genetic screen’)nd to tailor therapy accordingly.

. Activated protein C

Protein C has several effects in humans (Table 2). It is anti-nflammatory, anticoagulant and pro-fibrinolytic, counteract-ng some of the main pro-inflammatory pathophysiological

echanisms in sepsis. Levels of protein C are known to beow in sepsis.

Finally, in 2001 a trial of this novel therapeutic agentn sepsis reported a reduction in mortality [3]. The resultsrom this trial (the PROWESS trial) are summarised in

ro-fibrinolytic Inhibits PAI-1 (and indirectly via reduced thrombinand therefore TAFI)

NF, tumour necrosis factor; IL, interleukin; PAI-1, plasminogen activatornhibitor-1; TAFI, thrombin-activatable fibrinolysis inhibitor.

92 M. Hughes / International Journal of Antimicrobial Agents 28 (2006) 90–94

Table 3PROWESS results

Outcome Time to outcome CER EER RRR ARR (95% CI) NNT (95% CI)

Death Within 28 days 259/840 (0.308) 210/850 (0.247) 19.4 0.061 (0.023–0.107) 16 (9–44)Serious bleeding Within 28 days 17/840 (0.020) 30/850

CER, control event rate; EER, experimental event rate; RRR, relative risk reductnumber needed to treat; N.S., not significant.

Table 4Exclusion criteria in PROWESS

Morbid obesity, moribund; major surgery <12 h; head trauma, intracranialsurgery or stroke <3 months; previous intracranial lesion; epidural;congenital bleeding diathesis; gastrointestinal bleeding requiringintervention <6 weeks; trauma with bleeding risk; pancreatitis; chronicrenal failure; varices, cirrhosis, chronic jaundice or ascites; platelets<30 × 109/L; antithrombotic medication including >650 mg aspirin;

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altsfoataeHrowly approved the use of drotrecogin alfa activated in those

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recent deep venous thrombosis or pulmonary embolism;hypercoagulable condition; immunocompromised

ere not controlled. There were extensive exclusion crite-ia (Table 4), mainly designed to avoid excessive bleeding.he patients were heterogeneous and the groups were sim-

lar at baseline. There was a mixture of infecting organ-sms and sites of infection. Intention-to-treat analysis wassed and the primary end-point was 28-day mortality. Thereas full follow-up and good protocol adherence. A protocol

hange during the trial has been the source of much criticism,lthough it did not seem to cause a significant alteration inutcomes.

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able 5ubgroups by APACHE II score and treatment effect in PROWESS

opulation Death in group, n (%)

Drotrecogin Placebo

PACHE II quartiles1st (3–19) 33/218 (15.1) 26/215 (12.1)2nd (20–24) 49/218 (22.5) 57/222 (25.7)3rd (25–29) 48/204 (23.5) 58/162 (35.8)4th (30–53) 80/210 (38.1) 118/241 (49.0)

PACHE II halves1st/2nd quartiles 82/436 (18.8) 83/437 (19.0)3rd/4th quartiles 128/414 (30.9) 176/403 (43.7)

R, relative risk; 95% CI, 95% confidence interval.

able 6ubgroups by number of organ failures and response to treatment in PROWESS

opulation Death in group, n (%)

Drotrecogin Placebo

umber of organ dysfunctions1 42/216 (19.4) 43/203 (21.2)2 56/270 (20.7) 71/274 (25.9)3 56/214 (26.2) 75/217 (34.6)≥4 56/150 (37.3) 70/146 (47.9)

ingle vs. multiple organ dysfunction1 42/216 (19.4) 43/203 (21.2)≥2 168/634 (26.5) 216/637 (33.9)

R, relative risk; 95% CI, 95% confidence interval.

(0.035) −75% −0.015 (N.S.) −66 (N.S.)

ion; ARR, actual risk reduction; 95% CI, 95% confidence interval; NNT,

Drotrecogin alpha reduced 28-day mortality by 6.1%number needed to treat (NNT) of 16), a relative risk reductionf 19.4%. There was an increase in serious bleeding events3.5% versus 2.0% in the placebo group, a number needed toarm (NNH) from bleeding of 66).

The expense of the drug (ca. £5000 for a 70 kg patient)nd the implications for healthcare resources very quicklyed to post hoc analysis of subgroups in PROWESS. Clearly,hese analyses are fraught with difficulty. Despite the smallize of some of the important groups, they did suggest dif-erences in response to this agent depending on the severityf illness (Tables 5 and 6). The most impressive cut-off wasn APACHE II score of 25 (the APACHE score is designedo assess the risk of mortality based on Acute Physiologynd Chronic Health Evaluation). There was also a differ-nce between single organ failure and multiple organ failure.ence, the US Food and Drug Administration (FDA) nar-

ith an APACHE II ≥25, and the European Medicines Evalu-tion Agency (EMEA) agreed to its use in those with multiplergan failure.

Absolute risk reduction RR (95% CI)

–3.0 1.25 (0.27–2.02)3.2 0.88 (0.63–1.22)

12.3 0.66 (0.48–0.91)10.9 0.78 (0.63–0.96)

0.2 0.99 (0.75–1.30)12.8 0.71 (0.59–0.85)

Absolute risk reduction RR (95% CI)

1.8 0.92 (0.63–1.34)5.3 0.80 (0.59–1.08)8.4 0.76 (0.57–1.02)

10.6 0.78 (0.60–1.02)

1.8 0.92 (0.63–1.34)7.4 0.78 (0.66–0.93)

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This was not the end of the matter. The lack of long-termollow-up data available at the time (these data have noween published), the cost implications, the effectiveness (aspposed to efficacy) of drotrecogin alfa activated and the lackf a subgroup defined a priori who benefited from treatmentontinued to cause concern.

Over 4 years on, we have a clearer idea both of the groupsf patients who should be treated and perhaps what furtheresearch should be undertaken.

First of all we know who should definitely not be treated.reatment of children appeared to cause an increase in

ntracranial haemorrhage but no improvement in mortality14]. Patients with single organ failure and recent (<30 days)urgery had a mortality rate of 21% versus 14% for thosereated with placebo [15]. The Administration of Drotrecoginlfa (Activated) in Early Stage Severe Sepsis (ADDRESS)

tudy [16] attempted to recruit 11 000 patients with APACHEI scores <25 or single organ failure. It was stopped after 2640atients were enrolled because of lack of efficacy. With theROWESS data, this makes a convincing case that patientsith APACHE II <25 or single organ failure should not beffered drotrecogin alfa activated.

The group of patients for whom drotrecogin alfa acti-ated is currently indicated are those with a high risk ofeath from severe sepsis. This concept is discussed furtherelow.

However, there remains significant disquiet in some quar-ers [4,17]. First, with regard to efficacy (the beneficial effecteen when an intervention is made in a trial environment): theumber of patients in the subgroup with APACHE II ≥25 was17 and they were identified in a post hoc analysis. The groupho were identified a priori (all patients with severe sepsis

nd organ dysfunction) had no significant improvement inortality at any time up to 1 year, except at 28 days [18]. The

enefit at 1 year was restricted to those with an APACHE IIcore ≥25 (48% versus 59%; relative risk (RR), 0.73, 95%onfidence interval 0.60–0.88). The small number of patients317) in the ADDRESS study with APACHE II scores ≥25eans that it would be difficult to demonstrate any benefit,

ut in fact more patients given drotrecogin alfa activated diedhan patients given placebo (49/165 (30%) patients treatedith drotrecogin alfa activated died compared with 39/159

25%) treated with placebo).Second, with regard to effectiveness (the beneficial effect

een when an intervention is made outwith a trial): theajor concern for a drug with such serious potential side

ffects would be injudicious use of the agent outside itsicensed indications or use with insufficient regard for theontraindications. Two multicentre medical utilisation eval-ations [19] revealed that mortality was higher in thesenstitutions for each of the organ failure groups (tworgan failure, three organ failure and so on) than in the

ROWESS trial. This is not entirely surprising, since effec-

iveness is likely to be different from efficacy. Of inter-st, though, was that over one-third of patients (37%) hadontraindications that would have precluded their inclusion

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n PROWESS. These patients had a higher mortality thanhose without such contraindications (49.3% versus 40.7%;= 0.05).Third, with regard to safety: recently, an open-label,

ingle-arm, international study of drotrecogin alfa activatedas published [20]. The rate of severe haemorrhage wasreater than PROWESS (6.5% versus 3.5%). The authorsrgued that the higher post-infusion bleeding rate (whenrotrecogin alfa was no longer a plausible cause of the bleed-ng) suggested a higher background bleeding rate in this trial.he bleeding rate in the ADDRESS trial [16] was similar toROWESS.

. To whom should drotrecogin alfa be prescribed?ow to judge ‘risk of death’

The current guidance both from the FDA and from theMEA suggests that drotrecogin alfa should be given to thoseatients at high risk of death. There are different ways to judgehis risk in an individual. The most compelling factor in theubgroup analysis was APACHE II score (Table 5). Thoseith an APACHE II ≥25 is the only subgroup with outcomeenefit at 1 year. The APACHE II score is dependent on thehysiological derangement caused by the inflammatory (aspposed to the anti-inflammatory) response. The score is alsoncreased by chronic ill health, and in the PROWESS trialhose patients with chronic ill health did appear to gain mostenefit.

There are reservations regarding the use of such a score inhis context since it has only been validated for use in the first4 h of ICU stay and was not designed for individual prog-ostication. However, the practice of scoring for APACHE IIt the time of inclusion into a critical care trial is becomingommon and indeed was the way the system was used in allf the drotrecogin alfa trials. It is the method recommendedy the FDA.

The other major cut-off point in subgroup analysis is multi-le organ failure compared with single organ failure (Table 6).he improvement in risk reduction is not as large as the risk

eduction noted with an APACHE II score of 25 and the effects not seen at 1 year. Current guidance from the EMEA sug-ests this is the approach of choice.

In the absence of further trials, the two opposing camps arenlikely to be reconciled. Despite some ethical and practicaloncerns about a further placebo-controlled trial following aositive well-conducted trial in a large number of patients,epeating this trial in the group of patients for whom it isurrently indicated would resolve the issue.

It has recently been suggested [21] that the decision todminister drotrecogin alfa should be made by a senior clini-ian weighing benefit and risk. This is an interesting proposal,

ut the ability of doctors to predict risk of death is limited andependent on the disorder [22,23]. Despite concerns about these of more proscriptive criteria, there is some evidence baseo support them. There is no evidence to sustain the position

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hat clinical judgement is the best way to decide on risk ofeath in severe sepsis.

. Conclusion

Drotrecogin alfa activated (recombinant human activatedrotein C) may be the first product to change the coursef severe sepsis dramatically. This has not been provedeyond all reasonable doubt, but the evidence currentlyvailable demonstrates a considerable benefit when used forppropriate patients. It is expensive. The proposed mech-nism of action suggests that only a small proportion ofatients with sepsis (those with early sepsis and a substantialro-inflammatory response) will benefit. In the meantime,rotrecogin alfa (recombinant human activated protein C)hould be prescribed to patients fulfilling the following cri-eria:

severe sepsis of <48 h duration;a high risk of death (APACHE II ≥25 or multiple organfailure);being treated with best standard care by doctors experi-enced in the care of patients with sepsis;static or deteriorating clinical condition; ornone of the exclusion criteria in the PROWESS trial(Table 4).

eferences

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