Pharmacokinetics of Caspofungin

Acetate in Healthy Adult Cats

Jana Leshinsky BVSc MANZCVS MVS GradCertVetStud

Faculty of Science School of Veterinary Science

The University of Sydney

A thesis submitted in fulfilment of the requirements for the degree of ‘Master of Veterinary Clinical Studies’ 2018

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This is to certify that to the best of my knowledge; the content of this thesis is my

own work. This thesis has not been submitted for any degree or other purposes.

I certify that the intellectual content of this thesis is the product of my own work and

that all the assistance received in preparing this thesis and sources have been

acknowledged.

Jana Leshinsky

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Acknowledgements

Firstly, my sincere thanks go to my primary research supervisor, Professor Vanessa Barrs. Her patience and guidance in all aspects and the time and energy contributed to the project is truly appreciated.

Recognition must also go to my secondary research supervisor, Professor Julia Beatty and course work supervisor Dr Christine Griebsch for their endless support and encouragement throughout this process. Much appreciation to Professor Andrew McLachlan for his patience and willingness to provide pharmacokinetic tutorials at short notice, as well as helping with analysis of the experimental data. Acknowledgment and appreciation must also go to Associate Professor Ross Norris for analysing the samples and Dr David Foster for performing the modelling and simulations, as well as for providing insight into their respective fields of expertise.

Thank you to my colleagues at the University Veterinary Teaching Hospital Sydney for providing assistance in collecting samples and giving the cats lots of love and affection while in the clinic; it would not have been possible without their help.

I acknowledge the Australian Companion Animal Health Foundation 009/2015 who provided funding for this project, as well as Merck for supplying the internal standard.

Finally, to all my colleagues, friends and family, thank you for your support and

encouragement throughout this challenging and rewarding process – it was greatly

appreciated and I would have not been able to accomplish it without your support.

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Abstract

Background – Feline upper respiratory tract aspergillosis (URTA) is an emerging mycosis with worldwide distribution. It occurs in two forms – sino-orbital and sino- nasal. Sino-orbital aspergillosis (SOA) is an invasive fungal infection with high mortality due to difficulty in its treatment. SOA is commonly caused by cryptic species in Aspergillus section Fumigati that are often resistant to antifungal triazoles but are usually susceptible to caspofungin. Caspofungin, an echinocandin, is a lipopeptide antifungal that inhibits glucan synthase and prevents the synthesis of β- 1,3-glucans in the fungal cell wall. It is indicated for the treatment of refractory invasive aspergillosis in humans and has been used successfully in cats with sino- orbital aspergillosis. Pharmacokinetic data is lacking in cats and cannot be extrapolated from other studies as cats possess several genetic polymorphisms and unique metabolic pathways compared to other species.

Aims – To determine the pharmacokinetics of caspofungin acetate in healthy adult cats and determine an optimal dosing regimen that can be utilised in a clinical setting.

Materials and Methods – Eight adult healthy cats were administered caspofungin (1mg/kg IV) over 1hr (Day 1). Six cats subsequently received caspofungin (1mg/kg IV) daily for an additional 6 days (Day 2-7). Blood was collected at 0 h, 0.5 h, 0.75 h, 1 h, 1.25 h, 1.5 h, 2 h, 3 h, 6 h, 9 h, 12 h and 24 h after drug administration (Day 1), before the next dose (Days 2–7), and 24 h after final dosing (Day 8). Plasma caspofungin levels were determined using HPLC-tandem mass spectrometry. Nonlinear mixed-effects pharmacokinetic modelling and simulation was used to investigate caspofungin population pharmacokinetics and explore dosing regimens in cats using caspofungin minimum effective concentrations (MECs). In the final pharmacokinetic model an optimum maximum concentration (Cmax): MEC ratio of 10- 20 was used to guide caspofungin efficacy. Simulations were performed for dosing regimens (doses 0.25 – 1 mg/kg and 6 – 72 h dosing intervals) with and without the inclusion of a loading dose.

Results - Using a 1 mg/kg dose Cmax was 14.8 µg/mL, Cmax at steady state was 19.8

µg/mL, Cmin was 5 µg/mL and Cmax: MEC was >20 in 42.6% of cats after multiple doses. An optimal Cmax: MEC ratio was achieved in caspofungin simulations using iv

0.75 mg/kg q 24 h or 1 mg/kg q 72h. However, at 1 mg/kg q 72h, Cmin was < MEC (i.e. <1 μg/mL) in over 95% of the population. Using a daily dose of 0.75 mg/kg, the

Cmax: MEC was optimal and Cmin was > 2.5 μg/mL for more than 95% of the population. Using a loading dose of 1 mg/kg and daily dose of 0.75 mg/kg thereafter, the Cmax: MEC was optimal and Cmin was > 2.5 μg/mL for 98% of the population.

Conclusion and clinical importance – Mean plasma caspofungin concentrations were > 1.0 µg/ml for the duration of the 24 h sampling period, which exceeds the MIC effective against most Aspergillus species. Caspofungin given at 1 mg/kg IV q 24h is safe in cats. Based on the modelling data, a daily caspofungin dose of 0.75 mg/kg daily (with or without the use of a 1mg/kg loading dose) is likely to achieve target therapeutic concentrations, meet the proposed Cmax: MEC window and provide consistent exposure between doses.

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TABLE OF CONTENTS

Acknowledgements………………………………………………………………………..ii

Abstract……………………………………………………………………………………..iii

Table of contents.…………………………………………………………………………iv

List of figures……………………………………………………………………………...vi

List of tables…………………………………………………………………………….....vi

List of abbreviations...…………………………………………………………………...vii

Chapter 1. INTRODUCTION………………………………………………………………1

1.1 Introduction …………………………..………………………………………..1

1.2 Aim ..……………………………………………………………………………..4

Chapter 2. LITERATURE REVIEW……………………………………………………….5

2.1. Idiosyncrasies of feline drug metabolism………………………………..5

2.2. Overview of pathogenic fungi...…………………………………………..10

2.2.1. Pathogenic Aspergillus species…………………………………...12

2.2.2. Structure of filamentous fungi……………………………………..13

2.3. Antifungal therapy in small animal veterinary medicine……………..14

2.3.1. Amphotericin B……………………………………………………...16

2.3.2. Azole Antifungal drugs……………………………………………..17

2.3.2.1. Itraconazole……………………………………………….19

2.3.2.2. Voriconazole………………………………………………20

2.3.2.3. Posaconazole……………………………………………..20

2.3.3. Pyrimidines………………………………………………………….21

2.3.4. Echinocandins………………………………………………………22

2.3.4.1. Mechanisms of action……………………………………22 vi

2.3.4.2. Pharmacokinetics of echinocandins……………………23

2.3.4.3. Clinical efficacy of echinocandins………………………25

2.3.4.4. Adverse events associated with echinocandins………27

2.4. Future directions and novel antifungal therapies……………………..28

2.5. Conclusion……………………………………………………………………31

Chapter 3. MATERIALS AND METHODS……………………………………………..32

3.1. Animals………………………………………………………………………..32

3.2. Drug Administration ………………………………………………….…….32

3.3. Blood Collection ……………………………………………...... 33

3.4. Analytical Method …………………………………………………………..34

3.5. Non-compartmental Pharmacokinetic Analysis …………………..…..35

3.6. Population Pharmacokinetic Analysis and Dose Regimen Simulations ……………………………………………………………………….35

3.6.1. Methods Software .………………………………...... 35

3.6.2. General Modelling Strategy ……………………………………….36

3.6.3. Covariate Model Building ………………………………………….37

3.6.4. Model Evaluation …………………………………………………..37

3.6.5. Caspofungin Dose Regimen Simulations ……………………….38

Chapter 4. RESULTS……………………………………………………………………..39

4.1. Safety………………………………………………………………………….39

4.2. Non-compartmental Pharmacokinetic Analysis ………………………39

4.3. Pharmacokinetic Modelling ……………………………………………….41

4.4. Caspofungin Dose Regimen Simulations ……………………………...44

Chapter 5. DISCUSSION…………………………………………………………………49

REFERENCES……………………………………………………………………………..55

APPENDIX I………………………………………………………………………………..84 vii

Leshinsky, J., McLachlan, A., Foster, D. J. R., Norris, R., & Barrs, V. R. (2017). Pharmacokinetics of caspofungin acetate to guide optimal dosing in cats. PLoS ONE, 12(6), e0178783. http://doi.org/10.1371/journal.pone.0178783 ; Supplementary material included.

APPENDIX 2 ……………………………………………………………………………102

CANCIDAS â (Caspofungin acetate) Product Information (S-WPC-MK0991-IV-102016)

List of figures

Figure 2.1 Diagram of the azole structures……………………………………...... 19

Figure 4.1 Mean plasma concentration-time curve for intravenous (IV) caspofungin acetate (1 mg/kg)……………………………………………40

Figure 4.2 Mean plasma concentration-time curve for IV caspofungin acetate (1 mg/kg) after reaching steady state……………………………………….41

Figure 4.3 Visual predictive check plot for 1000 simulations of the final pharmacokinetic model and observed data……………………………..43

Figure 4.4 Plots of caspofungin concentration over time…………………………..46

Figure 4.5 Plots of caspofungin Cmax:MEC over time……………………………….47

Figure 4.6 Plots of caspofungin Cmax:MEC ratio over time utilising a 1mg/kg loading dose on day 1 followed by 0.75 mg/kg daily thereafter……….48

List of tables

Table 2.1 Pathogenic fungal species of companion animals……………………..11

Table 2.2 Spectrum of activity of antifungals against common fungi…………….15

Table 2.3 Summary of the susceptibility of some of the “cryptic” Aspergillus species within section Fumigati…………………………………………..16

Table 2.4 Pharmacokinetics of echinocandins in adult humans………………….24

Table 4.1 Pharmacokinetic parameters following administration of a single 1mg/kg dose of caspofungin acetate IV and after reaching steady state after consecutive daily dosing of 1mg/kg IV……………………………40

Table 4.2 Population pharmacokinetic parameters for cats………………………42 viii

Table 4.3 Summary of caspofungin model-independent pharmacokinetic parameters and exposure indices from simulations which result in

maximum % animals 10

List of abbreviations

5FC Flucytosine ABLC Amphotericin B lipid complex AMB Amphotericin B AFG Anidulafungin ATP Adenosine triphosphate AUC Area under the concentration time curve BVS Between subject variability CAS Caspofungin CBC Complete blood count CI Confidence interval CL Clearance FLC Fluconazole ISA Isavuconazole ITC Itraconazole IV Intravenous MEC Minimum effective concentration MFG Micafungin MIC Minimum inhibitory concentration NAT N-acetyltransferase OJB Objective function value PCR Polymerase chain reaction POS Posaconazole PPV Population parameter variability RVC Ravuconazole SNA Sino-nasal aspergillosis SOA Sino-orbital aspergillosis TPMT Thiopurine S-methyltransferase UGT Uridine diphosphate glucuronosyltransferases URTA Upper respiratory tract aspergillosis USG Urine specific gravity V Volume of distribution VPC Visual predictive check VRC Voriconazole 1

Chapter 1 INTRODUCTION

1.1 Introduction

Fungal infections are less common than bacterial infections in veterinary medicine [1-3]. Consequently, there are fewer anti-microbial drugs available for the treatment of mycoses. However, due to the increased incidence of systemic fungal infections in human medicine, there has been an increased drive to explore new anti-fungal agents since the 1990s. The incidence of systemic mycoses in veterinary medicine, seems to be similarly increasing [2]. Pinner, Teutsch (4) reported that by the late 1990s fungal infection was the seventh most common cause of infection-related death in humans. This increase has been attributed to immunosuppression from immunodeficiency virus infections, haematopoietic stem cell and organ transplants, chemotherapy and haematological malignancies [3, 5, 6]. The development of fungal infections in critically ill patients has also been described in advanced liver cirrhosis and chronic pulmonary obstructive disease [7]. Global warming, leading to extension in the geographical range of pathogenic fungi has been implicated in the increased prevalence of mycotic disease in mammals [8].

Feline upper respiratory tract aspergillosis (URTA) is an emerging infectious disease with worldwide distribution [9]. The first case of feline URTA was reported in Australia in 1982 [10] and over 60 cases have since been reported in the veterinary literature, particularly in the last 5 years [9-24]. There are two anatomical forms of URTA in cats, sino-orbital aspergillosis (SOA) and sino-nasal aspergillosis (SNA), which are the result of colonisation of the upper respiratory tract with Aspergillus spp. Significant morbidity in affected cats occurs as a result of subsequent inflammation and tissue destruction [9]. SNA in cats is usually non-invasive and confined to the nasal passages and paranasal sinuses, similar to disease seen in canine SNA, although there have been reports of invasive SNA occurring in cats [25, 26]. SOA accounts for more than 65% of cases of feline URTA. The nasal passages are the primary site of infection; however SOA represents an extension of the disease to involve the orbit resulting in the development of mass lesions that can also extend 2 into the nasopharynx [15, 25]. Purebred cats, in particular brachycephalic breeds of Persian lineage, are over represented in cases of URTA and appear to be at increased risk [15, 21, 25, 27, 28]. Although invasive fungal infections are most commonly described in immuno-compromised individuals, SOA is associated with disease in apparently immuno-competent cats and carries a more guarded prognosis compared to SNA [9].

Aspergillus species are ubiquitous saprophytic fungi that are found in soil, water and decaying vegetation [29]. Sporulation occurs in decaying matter, producing a large number of small conidia, which are aerosolised and disseminate through the air via wind currents. The respiratory tract of both humans and animals is thus exposed to Aspergillus conidia on a daily basis. Aspergillus fumigatus is the most common Aspergillus species encountered by humans and animals [29]. In dogs and cats, it is most frequently associated with SNA [9, 30, 31]. SOA, on the other hand, is caused by cryptic species of Aspergillus in section Fumigati [9, 31]. Cryptic species, such as those within the A. viridinutans complex, are closely related groups of species that are indistinguishable from each other by morphological methods and require molecular methods for identification [32, 33]. In order to accurately identify fungal pathogens in humans and animals, molecular methods of identification are recommended, as correct species identification may facilitate improved treatment outcomes in cases of aspergillosis [25, 29, 34, 35].

Novel fungal species, and in particular cryptic species, are emerging pathogens of humans and companion animals. Barrs, van Doorn (31) described a novel heterothallic species in Aspergillus section Fumigati, Aspergillus felis, that was isolated from human patients with invasive pulmonary aspergillosis, cats with URTA and a dog with disseminated invasive aspergillosis. A. fumigatus section Fumigati contains more than 63 species, many of which cannot be clearly distinguished morphologically from one another or from A. fumigatus [35, 36]. A. felis is a member of the A. viridinutans complex, which contains other species capable of causing illness in cats include A. udagawae, A. wyomingensis and A. parafelis [18, 23, 26].

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As well as causing invasive disease these fungal species are often refractory to conventional anti-fungal therapeutic regimens. A. felis isolates have high minimum inhibitory concentrations of antifungal triazoles [31]. Isolates of A. felis can also exhibit in vitro cross-resistance to itraconazole and voriconazole, and to itraconazole, voriconazole and posaconazole [31, 37]. Optimal treatment protocols for cats with SOA have not yet been identified and are currently based on treatment responses from a small number of affected cats who required systemic antifungal therapy for at least six months.

The aim of systemic anti-fungal therapy is to eradicate the fungal organism without causing patient impairment. However, many antifungal agents have a narrow therapeutic index and adverse reactions can be problematic. Each agent is unique in its therapeutic role, toxicity profile and drug interactions. To maximise effectiveness, it is important to understand the pharmacokinetic and pharmacodynamics profile of each drug [38]. Treatment failures may also be attributed to their immense plasma concentration variance within and among individuals [39]. Despite the development of novel antifungal agents for treatment of human aspergillosis, some patients remain difficult to treat. This can be a direct result of drug interactions, antifungal resistance, fungal misidentification or organ dysfunction preventing the use of some agents [40].

Most antifungal drugs used in small animal veterinary medicine are not approved for use in companion animal species. They are used off-label, with indications and dosing regimens often extrapolated from human medicine [41]. This approach is not optimal due to the species differences in pharmacokinetics and susceptibility to toxic effects. Cats, in particular, are deficient in several drug conjugation pathways and have unique drug metabolism and disposition compared to dogs and humans, as well as genetic polymorphisms, which increases their risk of toxicity and make dose extrapolation potentially dangerous [42].

Caspofungin is an echinocandin approved in many countries for use in human medicine for the treatment of invasive aspergillosis that is refractory to other 4 antifungal therapy [43]. Caspofungin has a unique mechanism of action compared to the traditional antifungal agents such as amphotericin B and azoles [38, 44]. As echinocandins exert their antifungal activity directly on the fungal cell wall, adverse effects in mammalian species are expected to be low [45]. Drug-drug interactions and enhanced toxicity due to echinocandins are of minimal risk compared to triazole antifungals [1, 41].

Caspofungin was well tolerated and effective in a cat with sino-orbital aspergillosis [9]. A German Shepherd with disseminated invasive aspergillosis caused by A. deflectus was treated with caspofungin for over a year with no adverse effects documented [46]. The pharmacokinetics of micafungin, another echinocandin, have been studied in mice, rats and dogs [47]. Recently, the pharmacokinetic profile of a novel long acting echinocandin (CD101, biafungin) was investigated in mice, rats, dogs, cynomolgus monkeys and chimpanzees [48]. However, pharmacokinetic studies of other echinocandins, including caspofungin, have not been described in either the cat or dog. Current dosing regimens are extrapolated from human treatment recommendations. The pharmacokinetics of caspofungin in healthy cats needs to be described before clinical investigation in cats with naturally occurring fungal disease can be performed.

This review presents a brief overview of feline drug metabolism and pathogenic Aspergillus species, in particular those responsible for feline URTA. Current knowledge on anti-fungal drugs, with an emphasis on the echinocandin class, is also presented.

1.2. Aim

The objective of this study was to determine the pharmacokinetics of caspofungin acetate in healthy adult cats and to devise an optimal dosing regimen for the treatment of aspergillosis in a clinical setting.

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Chapter 2 LITERATURE REVIEW

2.1 Idiosyncrasies of feline drug metabolism

There has been considerable research undertaken to improve our understanding of drug metabolism and disposition in cats compared to other species; particularly deficiencies in drug metabolism pathways [49-68]. Xenobiotic metabolism plays a central role in the therapeutic and toxic effects that these pharmaceutical compounds may possess. If a species lacks the preferred metabolic pathway for the drug, elimination times will be prolonged and alternate metabolite production will be difficult to predict [69]. The metabolic pathways that have been studied in cats include glucuronidation (uridine diphosphate glucuronosyltransferases (UGTs)), acetylation (N-acetyltransferase isoform (NATs)), methylation (thiopurine methyltransferase) and active transporters (ATP-binding cassettes). Court (42) reviewed the pharmacokinetic evidence of species differences for drug metabolism among cats, dogs and humans; drugs that undergo metabolic conjugation, including glucuronidation, sulfation or glycination, such as aspirin, propofol and paracetamol can be cleared more slowly in cats [42, 69].

Glucuronidation deficiency is one of the most widely appreciated pharmacological idiosyncrasies of cats. If glucuronide synthesis is the major pathway of inactivation and other pathways are less efficient, there may be toxic accumulation of substances [69]. The elimination of many drugs, toxins and endogenous compounds into urine and/or bile is facilitated by conjugation with glucuronic acid; this process is catalysed by UGT enzymes expressed in the liver, kidney and intestinal mucosa, which are the primary sites of drug metabolism. Glucuronidation deficiency in cats mainly affects compounds with a simple planar phenolic structure [55, 70]. In humans and some other species, it has been shown that these compounds are mainly metabolised in the liver by UGT1A6 and UGT1A9 [56]. There does not appear to be a related UGT1A isoform expressed by the feline liver, and a mutation in the UGT1A6 gene results in a non-functional protein [53, 57, 71]. Additionally, the human liver 6 expresses five different UGT1A isoforms whereas cats only express two different UGT1A isoforms [57].

UGT1A1 is conserved in all species as it is essential for the glucuronidation and clearance of bilirubin. UGT1A2, the other isoform expressed in the feline liver is believed to be related to human UGT1A3 and UGT1A4, which glucuronidate drugs containing carboxyclic acid and amines [57]. Deficiency in functioning UGT1A6, the UGT isoform primarily responsible for paracetamol metabolism, causes shifting of paracetamol metabolism to the less efficient sulfation pathway contributing to toxicity in cats [57]. Other UGT isoforms, such as UGT2B7 and UGT2B15, are important for glucuronidation of other drugs (Court 2013). van Beusekom, Fink-Gremmels (53) showed that cats are also deficient in UGT2B7 and this may account for reduced morphine glucuronidation in cats [42].

Benzoic acid poisoning and hepatotoxicity has been shown in cats. The likely result of their UGTB isoforms is an inability to glucuronidate benzoic acid and benzyl alcohol, which are frequently added to drugs as preservative [54, 69]. Cats are able to glycinate benzoic acid, although this is a much slower process than glucuronidation and toxic accumulation tends to occur [72]. Although deficient glucuronidation is often cited as the cause of drug toxicity or slowed clearance in cats, there is only direct evidence of paracetamol, chloramphenicol, clofibrate, morphine, orbifloxacin and valporate being poorly glucuronidated [50, 51, 56, 73-77].

The ATP-binding cassette (ABC) transporters are a group of transmembrane proteins that derive energy from ATP to transport various molecules across cell membranes [60]. These transporters modulate drug absorption, distribution and elimination according to their level of expression in the intestine, liver, kidney, and at biological barriers such as the blood-brain barrier [78]. There are four members of this group of transporters that are known to be responsible for transporting drugs commonly used in veterinary patients, including ABCB1 (MDR1 or P-glycoprotein), ABCC1 (multidrug resistance-related protein-1) and ABCC2 (multidrug resistance- 7 related protein-2). Some of the commonly used drugs in veterinary medicine that rely on ABC transporters include fluoroquinolones, loperamide and cimetidine [78].

Permeability glycoprotein (P-glycoprotein) has been shown to influence the pharmacokinetics of many substrates including macrocyclic lactones in mammals, by actively transporting absorbed substrates back across a variety of cell membranes [79]. P-glycoprotein is found in all mammalian species and is widely distributed in tissues. However, a defect in the gene that codes for P-glycoprotein can result in accumulation of substrates and compromise protective barriers such as the blood brain barrier, causing toxicity [80]. A defect in the MDR1 gene, which encodes for P- glycoprotein, is associated with ivermectin sensitivity in herding dogs [81, 82]. Although a similar defect has not yet been described in cats, there have been anecdotal reports of ivermectin toxicity in cats, which could be due to the same mechanism [82]. The tissue distribution of P-glycoprotein in cats has been investigated. Van Der Heyden, Chiers (66) found similar distribution of P- glycoprotein in the liver, colon, adrenals and brain to humans and canines; however, it is not equally distributed in the kidney. This may have implications for feline renal susceptibility to toxic substances.

Fluoroquinolone antibiotic toxicity in cats is associated with temporary or permanent blindness [83, 84]. Ramirez, Minch (61) suggested that this toxicity is due to deficiency in the ABCG2 transporter resulting in inefficient efflux of fluoroquinolones from the feline eye. Like other ABC transporters, ABCG2 pumps substrates out of the cell, thus protecting the host from toxicity. Four amino acid changes have been identified in the feline ABCG2, and it is unknown if a combination of these or a single amino change is responsible for the defective function of the ABCG2 transporter [60, 61].

Mealey (60) hypothesises that deficient ABCG2 function in cats could lead to shunting of paracetamol metabolism away from sulfation and towards cytochrome P450-mediated pathways, thus further contributing to paracetamol toxicity in cats rather than a pure deficiency in UGT1A6 [57]. Defective ABCG2 function in cats may 8 have other physiological consequences as have been demonstrated in ABCG2 knockout mice and humans with functional polymorphism of ABCG2 [60]. ABCG2 has a role in protecting erythrocytes from oxidative damage, thus their deficiency may also be responsible for drug-induced erythrocyte damage such as the development of Heinz bodies and/or methaemoglobinaemia with paracetamol [60].

Arylamine drugs, such as sulphonamide antibiotics, hydralazine and procainamide, are metabolised through the N-Acetylation pathway. N-acetylation is catalysed by the N-acetyltransferase enzymes NAT1 and NAT2. Canid species, including domestic dogs, lack both of the genes encoding these enzymes [68]. Cats lack NAT2 but still express NAT1 with lower enzyme activity than other species [49]. NAT2 deficiency has been associated with low acetylation in the feline liver of various sulphonamides and is also proposed to contribute to the mechanism of paracetamol toxicity n cats as well as dogs [42, 49]. N-acetylation is the major mechanism of metabolism of caspofungin acetate in humans and “pre-clinical” species (mice, rats, rabbits and monkeys), thus toxicity due to deranged acetylation in cat livers could be a limiting factor in its use [45, 85].

Cats are particularly sensitive to the myelosuppressive effects of thiopurine drugs such as azathioprine [62, 67, 86]. S-methylation by thiopurine S-methyltransferase (TPMT) is an important detoxification mechanism for several chemotherapeutic and immunosuppressive drugs, including azathioprine [42]. Salavaggione, Yang (62) demonstrated that feline erythrocytes have significantly lower TPMT activity, which is a reflection of low enzyme activity in other tissues, compared to humans and dogs. There was also large individual variation in the level of TMPT activity [62]. The low activity of thiopurine S-methyltransferase may help to explain the sensitivity of cats to thiopurine drugs [62]. The reason for low TMPT levels in cats is unknown; however, several polymorphisms in the coding sequence that affect enzyme protein levels and activity for the feline TMPT gene have been identified and could explain this finding [42, 62].

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Acetylsalicylic acid (aspirin) has been used to treat acute pain and inflammation and as an antithrombotic agent for decades in cats, dogs and humans. However, it is eliminated in cats at a much slower rate relative to other mammals and therefore can only be used at doses 2-4 x lower and frequencies 4-6 x longer in cats than dogs [42]. In experimental human studies it has been shown that acetylsalicylic acid is rapidly converted to salicylic acid in the circulation and excreted in urine unchanged or after conjugation with glucuronic acid or glycine [87]. Unlike humans and dogs, cats have been shown to be deficient in the conjugation of salicylate with glycine to form salicylurate, and this is the likely mechanism of slow elimination rather than deficient glucuronidation [42, 73]. Poor glycine conjugation may therefore be the underlying cause for the slow elimination of aspirin in cats.

The hepatic cytochrome P450 enzymes play an important role in hepatic drug detoxification and other life processes such as cholesterol metabolism, bile-acid biosynthesis, and vitamin D3 synthesis and metabolism [88, 89]. Mutations in human cytochrome P450 genes can cause inborn errors in metabolism [71, 88]. Cytochrome P450 subfamilies are important in phase one metabolism of drugs. CYP1A, 2C, 2D and 3A subfamilies are important for the oxidative metabolism of many drugs in animals [63, 71, 89-92]. The CYP450 pattern of cats has only been investigated recently; CYP2C (corresponding to CYP2C9 in humans) activity in cats is extremely low and there is a difference between male and female cats in CYP2D and 3A activities [63]. Shah, Sanda (63) also compared CYP1A clearance reactions and found that its activity in cats was much higher than in dogs and humans, implying faster elimination rates of CYP1A substrates (e.g. theophylline) in cats. CYPD2E and CYP2D6 have also been explored recently and their low activity in cats are thought to further contribute to feline sensitivity to drugs such as paracetamol and volatile anaesthetic agents as compared to dogs and humans [52, 58, 65]. Many of the drugs used in veterinary medicine for dogs and cats have the same dose regime, but do not take into account the differences in CYP450 metabolism. These preliminary studies of CYP-mediated drug metabolism in cats show that we should carefully assess known CYP substrates prior to extrapolating dose regimes from other species for their use in cats.

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Drug metabolism and disposition differences in cats are still largely unknown, although increasingly more evidence is arising to suggest that these differences are significant. Physiological differences in cats such as a simple, shorter digestive tract, which has a slower transit time and greater permeability, may also affect the absorption of drugs [93, 94] Therefore, it is important that pharmaceuticals undergo individual species pharmacokinetic and pharmacodynamic investigation prior to their widespread and long-term use.

2.2 Overview of Pathogenic Fungi

Fungi are eukaryotic organisms of the kingdom fungi [95, 96] with a definitive cell wall composed of chitin, a structural polysaccharide (N-actelyglucosamine) that is absent in vertebrates [96, 97]. Fungi can be classified as dermatophytes, yeasts, dimorphic pathogens, oelomycetes, zygomycetes, oomycota phaeohyphomyocetes and hyalohyphomycetes [96]. Table 2.1 contains a list of fungal species known to be responsible for disease in cats and dogs [41, 95, 96, 98-100].

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Table 2.1. Pathogenic fungal species of companion animals.

There are two groups of systemic mycoses: dimorphic and opportunistic. Dimorphic species are characterised by their divergent morphological structure under specific environmental conditions [95]. These fungal pathogens are able to overcome physiological and cellular defences of the normal host by changing their morphological form [96]. Dimorphic fungi have a variable geographical distribution; with the primary site of infection often being the respiratory system due to inhalation of conidia [96].

Opportunistic systemic mycoses occur most commonly in debilitated animals, whose normal defence mechanisms are impaired. Organisms within this group include yeasts, the zygomycetes, Aspergillus spp., the other hyalohyphomycetes and phaeohyphomyocetes. Phaeohyphomyocetes are fungal moulds that characteristically produce melanin-pigmented hyphal elements in tissue and in culture [101]. The hyalohyphomycetes are non-pigmented and are transparent or hyaline in tissues [101]. Both are ubiquitous saprophytic agents that often cause infection in immunocompromised hosts after traumatic implantation from the environment or via the respiratory system [99, 102]. Opportunistic fungal infections 12 causing disseminated disease, e.g. disseminated aspergillosis or mucormycosis have been reported in immune-compromised cats secondary to diabetes mellitus, viral infection (feline leukaemia virus, feline immunodeficiency virus, feline panleukopaenia virus), and in those treated with glucocorticoids or antibiotics [103, 104].

Localised infection, for example with Aspergillus spp. and Cryptococcus spp., particularly of the sinuses and nasal cavity occurs frequently in companion animals [100]. Other filamentous fungi implicated in feline rhinitis include Penicillium spp., Scedosporium apiospermum, Alternaria alternate and zygomycetes such as Pythium insidiosum [100, 105-107].

2.2.1 Pathogenic Aspergillus species

Aspergillus fumigatus is the most important pathogenic filamentous fungus in humans. Cryptic species from Aspergillus section Fumigati are involved in 3 to 6% of invasive aspergillosis cases [32]. The actual prevalence may be higher due to lack of recognition of cryptic species by conventional diagnostic approaches. Other pathogenic Aspergillus spp. been implicated in aspergillosis in humans include A. flavus, A. niger, A. terreus, A. versicolor, A. nidulans and their cryptic species, as well as A. calidoustus, [108].

The species within Aspergillus section Fumigati that are most commonly associated with clinical disease in humans, apart from A. fumigatus, are A. lentulus, A. udagawae, A. felis-clade, A. thermomutatus (Neosartorya pseudofischeri), A. novofumigatus, and A. hiratsukae [32, 34, 35, 109]. Feline URTA is most commonly due to infection by members of Aspergillus section Fumigati [25, 110]. A. fumigatus is commonly identified as the pathogen involved in feline SNA [13, 21, 25, 110, 111]. Feline SOA, on the other hand, is most commonly caused by cryptic species, which have a lower thermotolerance and different secondary metabolite profiles that may contribute to their virulence [112, 113]. In addition, these cryptic species often exhibit decreased susceptibility to triazole and other antifungals, and are associated with high mortality rates [32, 33, 35, 36, 113, 114]. 13

Antifungal susceptibility was tested for thirteen A. felis isolates from cats by Barrs, van Doorn (31). In these tests, after incubation at 38oC, caspofungin displayed a minimum effective concentration (MEC) of less than 0.06 µg/ml in all but one isolate, which was 2 µg/mL. The majority of isolates had minimum inhibitory concentrations (MICs) greater than 0.06 µg/mL of amphotericin B and the azoles tested; with voriconazole having 9/13 isolates having MICs greater than 1 µg/mL [31]. Further susceptibility testing carried out by Lyskova, Hubka (33) has further shown that isolates of A. felis have relatively high MICs for voriconazole, itraconazole and amphotericin B. The MICs for posaconazole, caspofungin, anidulafungin and micafungin are, however, very low and likely consistent with a more favourable clinical outcome [33].

2.2.2 Structure of filamentous fungi

Antifungal agents generally target various components of the cell wall, thus in order to understand the pharmacodynamics of these agents it is important to appreciate the components of the fungal cell wall. For example, echinocandins inhibit glucan synthase whereas triazoles inhibit the synthesis of ergosterol. Hyphal growth is the life force of filamentous fungi, and is important for all aspects of colonisation, reproduction, morphogenesis and recognition of environmental signals [115]. Hyphae are broadly distinguished as septate or aseptate [115].

Each hypha has a cell wall, which is an extracellular matrix made up of an outer layer of polysaccharides and glycoproteins, and an inner layer of carbohydrate polymers including chitin, glucans and galactomannan [97, 116, 117]. Chitin is a structural polysaccharide (N-acetylglucosamine) that is absent in vertebrates [96, 97]. The nuclear envelope is a porous double membrane containing abundant RNA [115]. Within the cell wall, the plasma membrane contains ergosterol, a cell membrane sterol that is frequently targeted by anti-fungal agents [95]. Ergosterol regulates permeability of the cell membrane and the activity of membrane-bound enzymes [118]. Chitin synthesis is stimulated by low ergosterol content and inhibited by high concentrations causing patchy chitin formation [118]. 1,3-β-D-glucan is an essential cell wall homopolysaccharide found in the carbohydrate layer of the fungal 14 cell wall. This carbohydrate layer also contains β-1,6-glucan and chitin [116, 117, 119].

1,3-β-D-glucans are not present in mammalian cell walls. 1,3-β-D-glucan synthase is a multi-subunit enzyme complex responsible for fungal cell wall synthesis, maintenance and remodelling [116]. Glucan synthase is a heteromeric enzyme complex comprising one large integral membrane protein, encoded by either the FKS1 or FKS2 genes, and one small subunit more loosely associated with the membrane, specified by Rho1 [120-122]. FKS1p is involved in cell wall remodelling and growth [120]. The FKS2 gene product is needed for sporulation [120-123]. Additional components, such as Pma1 have also been identified and may be involved in cell wall integrity [124].

2.3 Antifungal therapy in Small Animal Veterinary Medicine

Compared to antibacterial drugs the range of antifungal drugs is limited, particularly in veterinary medicine where the cost of newer, non-generic drugs is often prohibitive. Prior to the widespread availability of antifungal tri-azoles, amphotericin B was used for first-line treatment for invasive fungal infections in both human and veterinary medicine [1, 5]. The azole group of drugs have similar mechanisms of action and share other properties such as pharmacokinetics, tissue penetration and adverse effect profiles. Echinocandins are a fairly new class of antifungal that are gaining popularity in veterinary medicine. Table 2.2 is a schematic representation of the antifungals available for off-label use in companion animal veterinary medicine and their activity spectrums against some of the common fungal organisms extrapolated from available data [1, 2, 31, 34, 35, 38, 125-140]. Table 2.3 shows the susceptibility patterns of Aspergillus cryptic species in section Fumigati [32, 114, 141-145].

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Table 2.2. Spectrum of activity of antifungals against common fungi. The data has been extracted from the listed references [1, 2, 31, 34, 35, 38, 125-140].

(+) indicates activity against organism, (-) indicates no activity against organism, (±) indicates variable activity against organism. No entry indicates unknown activity. Antifungals: Amphotericin B (AMB), Fluconazole (FLC), Itraconazole (ITC), Voriconazole (VRC), Isavuconazole (ISA), Ravuconazole (RVC), Posaconazole (POS), Caspofungin (CAS), Micafungin (MFG), Anidulafungin (AFG), Flucytosine (5FC)

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Table 2.3. Summary of the susceptibility of some of the “cryptic” Aspergillus species within section Fumigati [33, 34, 40, 99, 126, 127, 129, 134, 146, 147].

Species Amphotericin B Voriconazole Posaconazole Itraconazole Caspofungin

A. viridinutans - - + - +

A. felis + ± ± ± +

A. thermomutatus + - + - +

A. udagawae ± ± + + +

A. hiratsukae + + + + +

A. lentulus - - ± - +/±

A. novofumigatus + - - - +

(-) indicates no activity against organism; (+) indicates activity against organism; (±) indicates variable activity against organism.

2.3.1. Amphotericin B

Amphotericin B (AMB), a polyene macrolide antibiotic produced from Streptomyces nodosus, was discovered in 1956. AMB forms micelles with fungal ergosterol, forming channels in the fungal membrane and altering cell permeability, allowing leakage of ions and cellular components from the fungi and resulting in cell death [1, 5, 148]. It also activates macrophages and enhances macrophage-killing capacity [5]. This immunomodulatory effect may explain its effectiveness against fungi lacking cell wall ergosterol, such as Pythium insidiosum [41].

AMB has been utilised as first line therapy for many systemic fungal infections in humans, dogs and cats [1]. Resistance to AMB is rare, although high MIC values have been observed for some moulds, including Aspergillus terreus and A. flavus, A. lentulus and A. fumigatiaffinis [35, 41, 141, 149-152]. This may be related to decreased cell wall ergosterol, however the mechanism is not well understood [153]. AMB is minimally absorbed from the gastrointestinal tract, and therefore requires intravenous administration. However, it causes cumulative nephrotoxicity due to direct toxicity to epithelial cell membranes, and renal vasoconstriction causing 17 reduced renal blood flow and glomerular filtration rate [1, 5, 41, 154]. New formulations of AMB were subsequently developed; liposomal preparation, lipid complex and colloidal dispersion with cholesterol sulphate [1]. These preparations are less nephrotoxic, however are considerably more expensive [1, 41].

While AMB formulations are commonly used, evidence-based research is lacking in veterinary medicine. According to Sykes and Papich (41), amphotericin B lipid complex (ABLC) is the most commonly used formulation in companion animal medicine for systemic mycoses. Disruption of lipid complexes by phospholipases occurs only at sites of inflammation or infection, leading to the release of AMB [1, 155]. In research dogs, ABLC was found to be eight to ten-times less nephrotoxic than conventional AMB; however, its toxicity profile in cats has not been determined [156]. Experimental studies in healthy beagle dogs showed six-times higher plasma concentrations of liposomal amphotericin B (AmBisome) than original AMB, no azotaemia and minimal renal effects on histopathology [157]. Barrs, Fliegner (9) reported the successful use of liposomal amphotericin (AmBisome) in cases of sino- orbital and sino-nasal aspergillosis in combination with itraconazole and posaconazole. This formulation was also reportedly used in another cat with sino- orbital aspergillosis which was refractory to azole treatment. The cat was euthanized one month after starting treatment due to seizures associated with intracranial extension of infection [15]. This evidence suggests that the new preparations of AMB have improved efficacy and a better safety profile; however, no pharmacological studies have been conducted in cats and therefore caution is still warranted in this species.

2.3.2. Azole antifungal drugs

Azole antifungals, similar to amphotericin B, also target ergosterol in the fungal cell wall. However, in contrast to AMB, azoles inhibit sterol 14α-demethylase, a cytochrome P450-dependent fungal enzyme involved in the synthesis of ergosterol from lanosterol [41]. This leads to ergosterol reduction and accumulation of anomalous and potentially lethal sterols in the cell membrane [1]. Most of the adverse effects and drug interactions with this class of drugs are a direct result of 18 cross-inhibition of mammalian cytochrome P450 enzymes [1, 41]. Azoles, imidazoles and triazoles, are a group of synthetic aromatic compounds that are structurally similar with a five-membered azole ring and a complex side chain [158]. Imidazoles are now largely used for topical use whereas triazoles are recommended for systemic disease. Imidazoles such as ketoconazole were previously used for systemic mycoses, due to good oral bioavailability, however this drug was deregistered in Australia and the US in 2013 due to its significant toxic effects in humans [1, 41, 159-161]. Cats are also highly susceptible to the side effects of ketoconazole; including hepatotoxicity and suppression of steroid hormone synthesis [162]. Triazoles are metabolised more slowly and have less impact on mammalian sterol synthesis than the imidazoles [41].

“Triazole”, as shown in Figure 2.1, refers to either one of a pair of isomeric chemical compounds with the molecular formula C2H3N3, having a five-membered ring of two carbon atoms and three nitrogen atoms [163]. The structural differences of the azole rings result in different binding affinities for the cytochrome P-450 (CYP) enzyme system [163]. The triazole antifungal drugs include itraconazole and fluconazole, as well as the newer drugs, voriconazole and posaconazole. Itraconazole

(C35H38Cl2N8O4) is an equimolar racemic mixture of four diastereoisomers (two enantiomeric pairs), each possessing three chiral centres, it is structurally similar to ketoconazole (C26H28Cl2N4O4) [163]. Itraconazole has three nitrogen atoms in its azole ring, which help with tissue penetration, prolong half-life and increase specificity for fungal enzymes [158]. Posaconazole (C37H42F2N8O4) is a synthetic structural analogue of itraconazole, with fluorine in place of a chlorine and a furan ring in place of the dioxolane ring [131, 163]. Voriconazole (C16H14F3N5O) was developed from fluconazole (C13H12F2N6O), a fluorinated bistriazole, by substituting the fluoropyrimidine ring for one of the azole groups and an added α- methyl group, which provides activity against Aspergillus spp. and other moulds; unlike fluconazole to which Aspergillus spp. are intrinsically resistant [135, 163, 164].

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Figure 2.1. Diagram of the azole structures

2.3.2.1 Itraconazole

Itraconazole is one of the most widely used azoles in both human and veterinary medicine. It is often used as first-line systemic treatment for Aspergillus spp. infection in dogs and cats [1, 41, 165-167]. The most common adverse effects associated with itraconazole are gastrointestinal signs and hepatotoxicity [1, 41, 165- 168]. Itraconazole is available in both capsules and oral suspension. The oral suspension has enhanced bioavailability in cats and requires a dose reduction compared to the capsule in order to minimise hepatotoxicity, 1-1.5mg/kg per day and 10mg/kg per day respectively [1, 41, 168]. Itraconazole, like ketoconazole, can inhibit the metabolism of other P450 cytochrome drugs and interfere with P-glycoprotein transport of ivermectin [1, 41]. 20

2.3.2.2 Voriconazole

Voriconazole is used for first-line treatment of invasive aspergillosis in humans [169]. It is also used to treat other mould infections, particularly if they are refractory to other treatments, including some Scedosporium spp., and Fusarium spp. [20, 41]. The major draw-back of voriconazole is its cost and neurological side effects, as well as gastrointestinal and hepatic effects. In humans, it has been reported to cause reversible photophobia and blurred vision, hallucinations and peripheral neuropathies [170-172]. Toxicity has not been documented in dogs, which may be due to its limited use associated with high cost of treatment [173-175]. Gastrointestinal signs were reported in a pharmacokinetic study of voriconazole in dogs [176]. Sykes and Papich (41) also describe inappetence, increased serum liver enzymes activities and CNS signs such as ataxia and staring, as well as tachypnoea and pyrexia in dogs. Cats appear to be highly sensitive to the effects of voriconazole with several case reports detailing adverse events such as reduced appetite, lethargy, hypokalaemia, arrhythmias, CNS signs including ataxia, hind limb paresis, blindness, staring off into space and vestibular signs, and in one case death [9, 20, 177, 178]. Voriconazole, like itraconazole and ketoconazole, also inhibits CYP3A and can therefore increase plasma concentrations of other drugs [179]. A recent pharmacokinetic study of voriconazole in cats, suggested that a dose of 12.5 mg/cat every 72 hours compared to 5 mg/kg twice daily, as for dogs and humans, may be safe and effective [180]. The authors, however, recommended close therapeutic drug monitoring in order to minimise adverse effects.

2.3.2.3 Posaconazole

Posaconazole is a promising antifungal agent for a number of refractory deep mycoses in animals. It is reported to have the lowest minimum inhibitory concentration against isolates of Aspergillus Section Fumigati of the available azoles and may be effective when other azole treatments have failed [35, 164, 181]. Posaconazole is highly protein bound (>98%) and undergoes hepatic metabolism with only minimal drug interaction via the cytochrome P450 enzyme pathways [131, 182]. Initial studies in various species have shown that its bioavailability is promoted by concurrent feeding of a fatty meal; however, alterations in gastric acidity do not 21 affect its absorption [138, 183, 184]. Posaconazole (Noxafil, Merck) is available in an oral suspension, delayed release tablets and as an intravenous injection; however, its availability in all forms is limited outside of the United States. Posaconazole is well tolerated after oral administration of suspension in cats, however mild liver enzyme elevations have been noted [9, 15, 19, 185-187].

Published data on the pharmacokinetics of posaconazole in cats has recently become available. This has shown that although it is well tolerated with minimal side effects, it has low oral bioavailability and as such, a greater oral dose is required to reach therapeutic serum levels, which may predispose to adverse effects [188]. It has also been shown to be well tolerated long term in critically-ill people with refractory invasive fungal infections [189, 190].

The metabolism of posaconazole is mediated by uridine diphosphate (UDP) – glucuronyltransferase (UGT) enzyme pathways, particularly UGT1A4 [191]. It is also a substrate and inhibitor of P-glycoprotein, however the role of gene polymorphism in the pharmacokinetics of posaconazole have not yet been explored [191]. Other newly available azoles, include ravuconazole and isavuconazole [192, 193]. Isavuconazole has shown promise for the treatment of invasive Aspergillus fumigatus when there is cross-resistance with other azoles [134, 192, 194].

2.3.3. Pyrimidines

The pyrimidines (5-FC), such as flucytosine, have been used to treat azole resistant infections of Cryptococcus spp. in cats and in Candida spp. infection [2]. Flucytosine is often used in combination with azoles or AMB as an effective treatment for cryptococcosis in cats and dogs [195, 196]. They are inactive against most Aspergillus spp. The drug interferes with DNA/RNA synthesis when it is deaminated into 5-fluorouracil within the fungal cell. Unfortunately, there is rapid development of resistance with this antifungal and it has significant side effects such as myelosuppression, gastrointestinal upset, acute hepatic injury, renal failure, hypersensitivity and central nervous system signs [2]. 22

2.3.4. Echinocandins

2.3.4.1 Mechanisms of action

Echinocandins are large semisynthetic lipopeptides which have been chemically modified from the natural products of fungi [123]. Drugs within this class include caspofungin acetate, micafungin and anidulafungin, which received FDA approval in 2002, 2005 and 2006, respectively. A novel long-acting echinocandin (CD101 IV) is currently in phase two trials and shows promise as a once-weekly outpatient treatment for systemic mycoses, including potentially resistant pathogens [48, 197]. Each echinocandin is structurally different and has been derived from different fungal species; caspofungin is derived from pneumocandin B of Glarea lazoyensis, and anidulafungin comes from echinocandin B0 of Aspergillus nidulans [123]. These differences account for the variation in their pharmacokinetic profiles and drug interactions, despite similar spectrums of activity [123]. Their specific anti-fungal activity is determined by the position and confirmation of the N-linked acyl lipid side chains of the cyclic hexapeptides. Caspofungin has a fatty acid side chain, whereas micafungin has a complex aromatic and anidulafungin has an alkoxytriphenyl side chain [123]. These side chains interact with the phospholipid bilayer of the fungal cell membrane.

Echinocandins inhibit glucan synthase within the fungal cell wall via non-competitive inhibition of 1,3,β-D-glucan synthase, and to a lesser extent the 1,6,β-D-glucan synthase within the fungal cell wall [1, 41, 119, 123]. Specifically, echinocandins target the FSK1 gene product that encodes for the Fks1p subunit within 1,3,β-D- glucan synthase [119, 123]. The precise binding site in regards to the enzyme complex is unknown. Inhibition of 1,3,β-D-glucan synthase leads to compromise of the osmotic integrity of the cell wall and cell lysis. A second mechanism of action is thought to be due to disruption of cell wall mannoproteins, allowing for greater immune system recognition [1]. Hohl, Feldmesser (198) also demonstrated that b- glucan exposure due to echinocandin-induced morphological hyphal changes increase the dectin-1-mediated inflammatory responses by macrophages.

Caspofungin is fungicidal against Candida spp and fungistatic against Aspergillus spp., Cryptococcus neoformans and zygomycetes are resistant [41, 123, 140, 199]. 23

Yeast forms of fungi are not susceptible as they contain α-glucan within the cell wall, which is not targeted by echinocandins [1, 41]. The activity of the echinocandins is largely dependent on the proportion of the fungal cell wall comprised of glucan, which varies between fungal species [123]. As single agent therapy, echinocandins have minimal effect on Fusarium spp., zygomycetes and Scedosporium spp., as there is limited glucan within the cell wall. However, utilising caspofungin in combination with amphotericin B has been successful in the treatment of zygomycosis in murine models, as well as human patients [200, 201]. Caspofungin also has an enhanced effect in the treatment of aspergillosis and candidiasis when combined with azoles; such as itraconazole, posaconazole and voriconazole [202- 209].

2.3.4.2 Pharmacokinetics of echinocandins

Due to their poor oral bioavailability (e.g. <0.2% for caspofungin) echinocandins are available as intravenous preparations only [85]. However, they are highly protein bound (e.g. 96-97% for caspofungin) allowing for good distribution into the lung, liver and spleen [85, 210]. Their high molecular weight, as well as level of protein binding limits their ability to penetrate the central nervous system and ocular fluid [210, 211]. Their urinary concentrations are low, and they are not metabolized or excreted via the renal system [85, 212, 213].

Echinocandins exhibit dose-dependent linear pharmacokinetics after intravenous administration in all studied species; including humans, rodents, lagomorphs, dogs, cynomolgus monkeys and chimpanzees. In humans and rats, studies have shown that they are taken up by the red blood cells (micafungin) and the liver (caspofungin and micafungin) following initial distribution and are slowly degraded primarily in the liver, as well as the adrenal glands and spleen [210]. Degradation of caspofungin to inactive metabolites occurs by peptide hydrolysis in these species, as opposed to oxidative reactions and N-acetylation [45, 85, 210, 214]. Catechol-O- methyltransferase pathways in the liver metabolise micafungin in rats and humans [123]. The degradation products of caspofungin in rats and humans are eliminated over a number of days via the biliary system [210, 215]. Anidulafungin is unique 24 among the echinocandins as it has been shown that it is eliminated almost exclusively by slow degradation in bile rather than by hepatic metabolism in rats [216].

As can be seen in Table 2.4, pharmacokinetic parameters including maximum concentration, elimination pathways, elimination half-life, volume of distribution and clearance rates are different for each echinocandin drug administered to the same host. Adult human data is provided for comparison, as comparable pharmacokinetic data for other mammalian species is lacking. Anidulafungin, for example, has a lower maximum concentration with longer half-life, as well as larger volume of distribution and faster clearance than caspofungin and micafungin. Steady-state concentrations for caspofungin are achieved significantly later than with micafungin [123, 217].

Table 2.4. Pharmacokinetics of echinocandins in adult humans

Variable Caspofungin Micafungin Anidulafungin

Cmax (mg/ml) 7.64 4.95 2.07-3.5

Bioavailability (%) <10 <10 2-7

T1/2 (h) 9-11 11-17 24-26

Vd (L/kg) 0.14 0.22-0.24 0.5

AUC (µg.h/mL) 87.9-114.8 111.3 44.4-53

Protein Binding (%) 96-97 99.8 >99

Clearance (ml/min/kg) 0.14-0.17 0.12-0.3 0.24

Time to Steady State 2 weeks 4-5 days 1 day*

Cmax = maximum concentration; T1/2 = elimination half-life; Vd = volume of distribution; AUC = area under the plasma concentration-time curve. Table modified from Chen, Slavin (123) and Kofla and Ruhnke (217). Data for 50mg single dose IV in an average 70kg adult. *steady state achieved after single loading dose (100mg IV).

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The echinocandins do not serve as major substrates, inducers or inhibitors of cytochrome P450 or the P-glycoprotein transport system [121]. Deranged P- glycoprotein in mammals increases the risk of toxicity due to active transportation of substrates back across cell membranes [79]. Drug-drug interactions and enhanced toxicity due to echinocandins is of minimal risk, compared to triazole antifungals that interact with P-glycoprotein [1, 41]. Sandhu, Lee (215) showed that the OATP-1B1 transporter pathway is responsible for hepatic uptake of caspofungin. This also transports bile and several other drugs such as rifampin and cyclosporine. The use of rifampin with caspofungin necessitates increasing the daily dosage, as it initially inhibits and then induces hepatic uptake of caspofungin by OATP-1B1, which decreases steady state concentrations [218, 219].

2.3.4.3 Clinical efficacy of echinocandins

The spectrum of activity of the echinocandins is best measured by determining the minimum effective concentration (MEC) rather than the MIC. As echinocandins are fungistatic against filamentous fungal organisms such as Aspergillus spp., their effectiveness is measured by the lowest drug concentration causing blunt attenuation of hyphal structures viewed microscopically, known as the MEC [123]. The MEC is the recommended endpoint for determining in vitro activity of caspofungin against Aspergillus spp., and has been adopted in more recent susceptibility studies [128, 199, 220-222]. Pfaller, Boyken (221) determined the in vitro activity of caspofungin, anidulafungin and micafungin against Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger and Aspergillus terreus isolates, of which 99% were inhibited by <0.06 μg/ml of antifungal drug. Alcazar-Fuoli, Mellado (35) also showed that all tested strains of Aspergillus section Fumigati, are susceptible to both caspofungin and micafungin (MECs ≤1.3μg/ml). Echinocandin susceptibility is determined utilising CLSI and EUCAST reference susceptibility broth microdilution (BMD) methods [221, 223].

Echinocandin resistance is an emerging risk factor for treatment failure in human patients affected by aspergillosis, although it is considered a rare phenomenon. Similar to the mechanism of azole resistance; echinocandin resistance of moulds 26 has been associated with point mutations in hot spots of the FKS1 gene encoding the major subunit of 1,3-β-D-glucan synthase [224]. Resistance to caspofungin has been described for clinical isolates of Candida, and more recently, breakthrough infection with Aspergillus spp. with elevated MEC have also been reported in human patients [225, 226]. Laboratory manipulated strains of Aspergillus fumigatus with mutations in the ECM33 gene, encoding cell wall proteins important for fungal cell wall organisation, have been shown to be intrinsically resistant to caspofungin and are hypervirulent in mice [227]. Scientific studies surrounding antifungal resistance concentrate on Aspergillus fumigatus as it is the most common isolate causing human infection. There have also been clinical reports of resistance to echinocandins in humans infected with A. fumigatus and A. flavus previously exposed to this class of drugs, as well as breakthrough infections [226, 228]. Clinical resistance is multifactorial and attributed to host factors, virulence factors of the pathogen, and/or the pharmacodynamics and pharmacokinetics of antifungal drugs [153, 229, 230]. Cryptic species in Aspergillus section Fumigati, many of which are emerging pathogens, require further investigation as many are intrinsically resistant to one or more antifungal drugs [35].

Various models have looked at how best to describe the clinical efficacy of echinocandins and predict clinical outcome. Parameters examined include percentage of time greater than the MEC, 96-h area under the plasma concentration curve to MEC ratio, and peak (Cmax) in plasma to MEC ratio [231-235]. Andes, Marchillo (232) found that the peak concentration to MEC ratio was the most appropriate tool to monitor therapeutic dosing in a murine model of candidiasis treated with aminocandin. However, Li, Sun (233) found that none of these pharmacokinetic parameters were significantly correlated with outcomes in paediatric human patients with aspergillosis or candidiasis that were treated with caspofungin. In a murine model of invasive pulmonary aspergillosis in which caspofungin dose escalation and dose fractionation were evaluated, Cmax: MEC ratio was the parameter most strongly associated with reduced fungal burden with an optimal value of 10-20. Mice dosed with 1 mg/kg daily or 2 mg/kg every second day had a significantly lower fungal burdens than mice dosed with 1 mg/kg every 6 hours [231].

At higher doses of 4 mg/kg, correlating to a Cmax: MEC > 20 there was a paradoxical 27 effect on fungal growth resulting in increased burdens in mice. Although not proven in clinical trials, the paradoxical growth effect of caspofungin at Cmax: MEC > 20 in vivo has led to concerns about possible reduced clinical efficacy using dosing regimens where the Cmax greatly exceeds the MEC [231]. However, the in vivo significance of this is yet to be determined, as the doses exhibiting this effect exceed clinical recommendations.

In humans, caspofungin is indicated for treatment of invasive aspergillosis that is refractory to other antifungal therapy [7, 116, 121, 236-240]. The echinocandins do not appear to have cross-resistance to other antifungal agents and are generally effective against azole-resistant moulds and Candida spp. [116, 241]. Caspofungin is also recommended for use in patients that are unable to tolerate other antifungals due to hepatic or renal insufficiency [41]. Caspofungin is also approved as empirical therapy for presumed Candida spp. or Aspergillus spp. infections in febrile neutropenic patients [116].

2.3.4.4 Adverse events associated with echinocandins

As echinocandins exert their antifungal activity on the fungal cell wall their adverse side effects in mammalian species are expected to be low [45]. Adverse events reported in humans are minimal and include fever, gastrointestinal signs, phlebitis and headache [1, 41, 214]. Histamine-mediated signs including anaphylaxis, rashes, facial swelling, pruritus, bronchospasm and cardiac arrhythmias have been reported in humans during caspofungin infusion but are rare [208, 242]. Echinocandin induced cardiomyopathies due to focal direct myocyte injury in human patients and experimental rat models receiving echinocandins via a central line have also been rarely reported [243, 244]. Manifestations of acute anaphylaxis are species- dependent and directly related to the locations of the largest population of mast cells, which are the heart and lungs in humans, gastrointestinal tract and liver in dogs, and lungs in cats [245-252]. Thus, the establishment of a safety profile of echinocandins in cats is a crucial aspect of determining their pharmacokinetic profile.

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Caspofungin administered in excessive doses was shown to cause hepatic necrosis in one pre-clinical trial in humans, and subsequent studies have reported a low incidence of serious hepatoxicity [123, 253]. All echinocandins can cause liver enzyme elevations, however only micafungin causes focal alteration in hepatocytes and can cause hepatocellular tumours [254]. Discontinuation of micafungin is recommended if liver enzyme elevations are documented [254]. Mistry, Migoya (214) concluded that no dosage reduction is necessary in human patients with mild hepatic disease when treated with caspofungin, however recommended a 25% dose reduction in moderate hepatic disease.

Caspofungin, administered at 1mg/kg once daily IV, was well tolerated and effective in a cat with sino-orbital aspergillosis caused by A. felis [9]. A German Shepherd with A. deflectus infection was treated with caspofungin for over a year with no adverse effects attributable to its use [46]. The same dog also received anidulafungin upon clinical relapse, however developed a severe diffuse urticarial reaction [46]. Micafungin, in combination with ABLC was also trialled in this dog but there was no clinical response [46]. High-dose anidulafungin has been associated with the development of pulmonary oedema in neutropenic rabbits with pulmonary aspergillosis, although no adverse effects were reported using recommended doses [255]. Studies performed in rabbit models of invasive aspergillosis have also shown that neutropenia leads to significantly slower clearance of antifungals and higher plasma concentrations at the end of dosing intervals compared to healthy subjects, which may predispose to the paradoxical growth effect or toxicity [234]. A cat with sino-orbital aspergillosis due to A. udagawae was treated with a combination of AMB and micafungin and no adverse effects were reported [18]. Among companion animals, the pharmacokinetics of micafungin and the novel echinocandin CD101 (biafungin), but not caspofungin, have been determined in dogs, but no echinocandin pharmacokinetics have been evaluated in cats [47, 48].

2.4. Future directions and novel anti-fungal therapies

Despite the development of novel antifungals, fungal infections in some patients (including humans, cats and dogs) remain difficult to treat. Echinocandins are a 29 promising antifungal class for patients with aspergillosis refractory to other treatments. In particular, they exhibit excellent clinical efficacy when combined with other drug classes and have a favourable toxicity profile. However, development of other drugs with novel fungal targets is still required. The cost and time associated with research and development of novel anti-microbials often delays their clinical use. Criteria used to select optimal new antifungal drugs include potency when compared to AMB, safety compared to fluconazole, antifungal activity in vitro and in vivo, as well as differences in mode of activity [256].

The emergence of resistance to commonly used anti-fungal drugs has also spurred the requirement for the development of novel agents, as well as a better understanding of the ones currently in use. Potential novel therapies include pradimicins-benanomicins, nikkomycins, sordarins, cationic peptides, polyaminocarboxylates [256, 257]. A number of compounds with unidentified mechanisms of action are also being investigated which include dication-substituted carbazoles, furans, benzimidazoles, glycyrrhizin and a cyclic beta-amino acid related to cispentacin [256]. Pradimicins-benanomicins bind to cell wall mannoproteins, through calcium dependence, and have been shown to cause lysis and cell death via osmosis in animal models of aspergillosis [256]. In human trials this class of antifungal caused unacceptable hepatotoxicity, although future development with a better toxicity profile may allow for clinical use. Nikkomycins are competitive chitin synthase inhibitors. Other chitin inhibitors such as recombinant human chitinase also have efficacy in animal models of aspergillosis, but have greater synergistic activity with other antifungal compounds such as AMB [256]. Allylamines and thiocarbamates, including terbinafine, are reversible, non-competitive inhibitors of squalene epoxidase, which converts squalene to lanosterol and therefore prevents the production of ergosterol [256]. Although terbinafine has long been available as an anti-fungal agent, its usefulness as a single agent is limited. Further development of this class may also provide better in vivo effectiveness for aspergillosis. Sordarins have a novel mechanism of action, inhibiting protein synthesis in pathogenic fungi, and has synergistic effect against Aspergillus spp. and Scedosporium spp. when combined with AMB and azoles [256]. The cationic peptides have antifungal activity against a number of pathogenic fungi, including Aspergillus spp.; however their 30 clinical applicability remains to be determined [256]. Polyaminocarboxylates exert their effect by interfering with fungal zinc metabolism [257]. Another novel class of glucan synthase inhibitors, triterpenoid antifungals have also been developed, with broad in vitro and in vivo activity against a broad spectrum of Aspergillus [258].

Invasive aspergillosis in humans is treated with a combination of antifungal drug therapy and immune augmentation strategies, as disease occurs most commonly in immune-compromised hosts. This approach could be employed in veterinary medicine for patients with known immune-deficiencies. Another approach may be to further boost the immune system of immune-competent patients, for example cats with sino-orbital aspergillosis secondary to A. felis, in order to enhance fungal clearance. Current strategies for enhancing immune-competence in humans include improving phagocytic function, providing cytokines and stimulating innate pathogen recognition pathways. For example, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony stimulating factor have been shown to increase fungicidal activity of phagocytes in vitro against Aspergillus spp. [259, 260]. In cases of invasive aspergillosis caused by Aspergillus fumigatus, GM-CSF may be recommended in neutropenic patients as host defence depends on both neutrophil and macrophage function [259]. Recombinant interferon γ augments innate and Th1-dependent immunity contributing to host defence against aspergillosis, however its use has not been validated and is limited to a small number of case reports in the human literature [259]. The stimulation of toll-like receptors (TLRs) can activate specialized anti-fungal effector functions and inflammatory responses. The activation of TLR-4 in neutrophils is a proposed secondary mechanism of action of liposomal AMB in the treatment of Aspergillus fumigatus [261].

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2.5. Conclusion

Despite the introduction of a number of novel antifungal treatments since the turn of the century, the prognosis for animals with systemic fungal infections, especially filamentous fungi, remains poor. Cats are deficient in numerous drug conjugation pathways, which potentially limits the range of drugs that can be utilised. Defective glucuronidation of phenolic substances, such as paracetamol, is the best-understood conjugation defect in cats. It has also been demonstrated that cats are deficient in other conjugation pathways, such as N-acetylation by NAT2, S-methylation by TPMT and active transporters (ATP-binding cassettes). These deficiencies can lead to slow elimination times and toxic accumulation of drugs. Thus, in cats, pharmacokinetic and safety profiles cannot necessarily be directly extrapolated from other species for drugs that are metabolised by these variable pathways.

Antifungal therapy must be tailored to the individual patient and organism involved [2]. Pharmacological variables contribute significantly to the overall therapeutic outcome, including the mechanism of drug action, route of administration, absorption and side effects. There is a large amount of work that still needs to be done to better understand the molecular causes of pharmacokinetic differences in cats compared to other species. As new therapies emerge for the treatment of systemic fungal infections, it is important to establish the metabolism, bioavailability, elimination routes and toxic effects in cats to optimise therapeutic outcomes.

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Chapter 3 MATERIALS AND METHODS

3.1. Animals

Eight clinically healthy adult domestic shorthair cats were used in this study weighing 4.4 ± 0.56 kg. The mean age of the cats was 3.94 ± 1.84 years. There were four desexed female cats and four desexed male cats. Results of complete blood count (CBC), serum biochemical analyses and urine specific gravity (USG) performed 5 days prior to the study were within reference values for the commercial laboratory (IDEXX Laboratories, Inc). The cats were acclimatized to the clinical environment for three days prior to the start of the study and were housed individually in a dedicated ward of the Valentine Charlton Cat Centre located at the University Veterinary Teaching Hospital - Sydney. The room was environmentally controlled (room temperature (19 – 21 oC), humidity (30-50%), 12-hour light/dark cycle). The cats were fed a commercial dry and wet food diet and had water ad libitum. A veterinary examination was performed upon arrival, and cats were handled every four hours during the acclimatization period. During the study period a full physical examination was performed twice daily, as well as visual examination (for food, water, eating, drinking, urination and defecation) every four hours. The study was approved by the Animal Ethics Committee (AEC) of The University of Sydney (Approval no. 2015/775, 6th March, 2015).

3.2. Drug Administration

For blood collection, a 20-gauge 8 cm triple-lumen central venous catheter (MILACATHÒ, MILA International, Inc., Kentucky, USA) was aseptically inserted into the jugular vein using a modified Seldinger technique, and secured with a Kitty Kollar®. An Elizabethan collar was also placed to prevent the cats removing the catheter. The cats were sedated with IV medetomidine (10 μg/kg; Domitor®, Pfizer Animal Health Australia, West Ryde, NSW) and butorphanol (0.1 mg/kg; Ilium Butorgesic®, Troy Laboratories Pty Limited, Glendenning, NSW) for jugular catheter placement twenty-four hours prior to starting the study. This dose was repeated after 20 min if no effect was seen, and alfaxalone (0.5 mg/kg; AlfaxanÒ, Jurox, 33

Rutherford, NSW) was administered if the desired level of sedation was not achieved after a further 20 min. For IV drug administration, a peripheral catheter (22G IV radiopaque catheter; OPTIVA®, Smiths Medical International Ltd, Lanchashire, UK) was placed into the cephalic vein with a T-connector (T-Connector with Spin Lock, Codan US Corporation, Santa Ana, CA) extension.

The treatment consisted of caspofungin acetate (Cancidas®, Merck Sharp & Dohme (Australia) PTY Limited, South Granville, NSW) for IV infusion (1 mg/kg), 0.2 mg/mL with sterile saline solution (0.9% sodium chloride, Abbott Laboratories, Abbott Park, IL), and given as an infusion over one hour. Upon completion of the infusion 2 mL of sterile saline was flushed through the line. In six cats (3 male neutered, 3 female neutered) drug administration was repeated for a further 6 days at the same time each day for the multi-dose component of the study.

3.3. Blood Collection

Blood Samples (1.0 mL) were taken with 3 mL syringes (Becton Dickison, North Ryde, NSW) via the catheter in the jugular vein immediately before drug administration (time 0), and after IV administration at 0.5, 0.75, 1, 1.25, 1.5, 2, 3, 6, 9, 12, and 24 h. Blood samples were immediately placed in lithium heparin-coated tubes (Sarstedt Australia Pty. Ltd., Mawson Lakes, SA). At the time of each sample collection, 1 mL of blood was withdrawn into a heparinised syringe to remove the saline solution out of the extension system, another 1.3 mL was collected as sample, the withdrawn blood was then replaced, and the system was flushed with 0.5 mL of 0.9% sterile saline. The central venous catheter was flushed with heparinised saline solution (0.5 mL) every 4 h between subsequent blood sample collections. Samples were centrifuged at 12, 000 xg for 5 min (IDEXX StatSpin VT Centrifuge, IDEXX Laboratories Inc.) within 10 min of collection, and plasma was removed with disposable polyethylene transfer pipettes and stored in micro tubes (Sarstedt Australia Pty. Ltd., Mawson Lakes, SA), labelled according to collection time, at -80 °C until assay.

34

In six cats this blood sampling technique was repeated on day 7 of the study, after a further 6 days of dosing at 1 mg/kg q24 h. A blood sample (1.0 mL) was also collected prior to daily infusion (48, 72, 96 and 120 h). A further 2 mL of blood was collected at 24 h post final infusion for repeat biochemical analysis.

3.4. Analytical Method

Assay of caspofungin in feline plasma samples was undertaken in the Clinical Pharmacology Division, SydPath, St Vincent’s Hospital, Sydney, using high performance liquid chromatography (HPLC) tandem mass spectrometry.

Blood samples were prepared for assay using protein precipitation by adding methanol (80 µL) to 20 µL of calibrators, controls (drug free) and unknown samples using a stable isotope of caspofungin as internal standard (in methanol). The analytical reference standard was supplied by Merck Sharp & Dohme (Whitehouse Station, New Jersey 08889, USA). Chromatography and analysis was performed on a Shimadzu UPLC coupled to an 8050 tandem mass spectrometer in positive ion ESI mode, monitoring the transition monitoring the transition 547.6 > 538.5 and internal standard transition 549.5> 86.2. A Waters BEH C18 column was used with gradient elution with mobile phase A (water containing 0.1 % formic acid) and mobile Phase B (acetonitrile containing 0.1 % formic acid). The mobile phase gradient involved 100% A for 0.5 mi and a linear gradient to 0% A by 4.5 min. After raising the gradient to 100% B at 4.5 min it was held there for 0.5 min before returning to initial conditions for a further 1 min, making a total run time of 6 min.

The method was validated in accordance with the US FDA Guidelines for industry for bioanalytical method validation utilising pooled plasma from six untreated cats with known concentrations of caspofungin reference standard [262]. Linearity was demonstrated from 0.1 to 10.0 µg/mL.

Data for demonstration of accuracy and precision (four replicates on three occasions of spiked controls at 0.2 and 9.0 µg/mL) showed total precision of 12.0 and 4.3 % 35

(expressed as co-efficient of variation) respectively. Accuracy was 103.4 and 96.7 % respectively. Carry-over was demonstrated to be no more than 1.0 % of the lowest standard in blank matrix following injection of the highest standard (10.0 µg/mL).

3.5. Non-compartmental Pharmacokinetic Analysis

Non-compartmental analysis was performed using the observed plasma caspofungin concentration-time data in Microsoft Excel. The area under the plasma concentration–time curve to the final measured concentration value (AUC0-t) was determined using the trapezoidal rule [263]. The elimination rate constant (kel) was calculated using the log-linear slope of the terminal portion of the concentration–time profile [263]. The AUC was extrapolated to infinity (AUC0-∞) using the last observed concentration divided by kel [263]. The elimination half-life (t1/2) was calculated as ln2/kel. The clearance (CL) was calculated as CL = (F*Dose)/AUC0-∞ ,where F = 1 due to intravenous administration, and volume of distribution (V) was estimated using the equation V = CL/kel. At steady-state, the area under the plasma concentration–time curve over the dosing interval (AUCss,0-τ, τ = 24 h) was calculated using the trapezoidal rule. The accumulation ratio was determined using the equation AUCss,0-τ / AUC0-t. Results of pharmacokinetic analyses were reported as the median and standard deviation.

3.6. Population Pharmacokinetic Analysis and Dose Regimen Simulations1

3.6.1. Methods Software

Pharmacokinetic model development employed nonlinear mixed-effects modelling using NONMEM version 7.3 [264], with the Wings for NONMEM 7.3 interface (http://wfn.sourceforge.net) and IFort compiler. Modelling was performed using a Dell

1 Population pharmacokinetic analysis, modelling and simulations were performed by Assoc. Prof. David Foster, PhD. School of Pharmacy and Medical Sciences, Australian Centre for Pharmacometrics, Sansom Institute for Health Research, University of South Australia, Adelaide, South Australia, Australia. 36

PowerEdge R910 server with 4 by 10 core Xeon 2.26-Ghz processors running Windows Server 2008 R2 Enterprise 64- bit software. Data manipulation and post- run processing of NONMEM output was conducted using the R data analysis language (Version 3.2.2) and the packages ggplot2, GGally, foreign, tidyr, Hmisc, gdata, doBy, plyr, grid, stringr with associated dependencies [264-275].

3.6.2. General Modelling Strategy

The base pharmacokinetic model was developed in a step-wise manner. Pharmacokinetic models were coded using the built-in ADVAN subroutines of NONMEM. Linear kinetic models with 1 and 2 compartments were evaluated. The First Order Conditional Estimation (FOCE) method was used to fit models. The base model was selected on the basis of mechanistic plausibility, visual inspection of goodness-of-fit diagnostic plots, the precision of parameter estimates (se%<30% for fixed, <50% for random effects parameters), and the lowest value of the Akaike’s information criterion (AIC; Equation 1) in accordance with the number of parameters and the final NONMEM derived objective function value (OBJ). The base model was also required to pass the covariance step.

AIC = OBJ + 2 ∗ number of parameters Equation 1

Unless stated otherwise, population parameter variability (PPV) was represented using an exponential error model (Equation 2):

P = TVP ∗ � Equation 2

th where Pj is the individual value for the parameter in the j individual, TVP is the typical population value of P and ηj is an independent random variable with a mean of zero and variance ω2. PPV was systematically examined on each fixed-effect parameter. Models with and without covariance for P were investigated using the OMEGA BLOCK functionality of NONMEM.

A combined proportional (θprop) and additive (θadd) residual unexplained error model of the caspofungin concentrations (C) was used, where estimation of a THETA was employed and epsilon was fixed to zero (Equation 3): 37

� = � + θ ∗ � + θ Equation 3

th th Cij is the i concentration measured in the j individual, � is the model predicted Cij, and θ and θ are parameters representing the proportional and additive residual error, respectively. The influence of the additive component of the error was also examined by a comparison with a proportional only error model.

All base models were investigated with allometric scaling for total body weight (TBW) referenced to 4 kg and an exponent fixed of 0.75 for clearance parameters and 1 for volumes, although during base model development models was explored without allometric scaling on any parameter [276].

3.6.3. Covariate Model Building

The base model was screened for the influence of cat sex, guided by plots of individual parameter random effects versus sex if shrinkage was low. Sex was only retained in the model if there was a significant improvement in OBJ at the p<0.01 level.

3.6.4. Model Evaluation

Visual Predictive Checks (VPC) were used to assess the appropriateness of the candidate base and final models, faceted for any included covariates as appropriate. For the VPC’s, the median, 5th and 95th percentiles of the prediction-corrected observations were compared against the empirical 95% confidence intervals (CI) of the median, 5th and 95th percentiles of caspofungin concentrations from 1000 simulations of the original dataset. The predictive performance of the model was considered acceptable if the median, 5th and 95th percentile of the prediction- corrected observed data lay inside the CI’s of the prediction-corrected simulated data for the majority of the time.

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3.6.5. Caspofungin Dose Regimen Simulations

The final pharmacokinetic model for caspofungin in cats was used to perform dosing simulations designed to examine Cmax: minimum effective concentration (MEC) ratios following intravenous infusions of caspofungin. Simulations were performed utilising total drug in plasma rather than unbound drug, as published data available for MEC values have only been reported for the total drug. Based on data from a murine model of invasive pulmonary aspergillosis an optimum Cmax: MEC ratio of 10-20 was chosen as a marker of caspofungin dose efficacy [231]. Simulations were performed for dosing regimens of 0.25 mg/kg at 6 h intervals; 0.5 mg/kg at 6 h, 12 h and 24 h dosing intervals; 0.75 mg/kg and 1 mg/kg at 24 h, 48 h and 72 intervals; 2 mg/kg at 48 h and 72 h dosing intervals. Loading dose regimen simulations were also considered based upon the results of the constant dose regimens. A MEC value of 1 µg/mL was chosen based on the epidemiological cut-off value (ECV) of caspofungin for wild type A. fumigatus sens. str [277] and on antifungal susceptibility MEC data on clinical human and feline isolates of A. felis [31] and A. udagawae [34, 144, 278]. The duration of infusion was adjusted to 1, 2 and 4 h in the 1 mg/kg and 2 mg/kg simulations. Concentrations were simulated at 0.1 h intervals for 1000 standard 4 kg cats for each dose regimen for a total of 7 days of dosing. The percentage of animals with a Cmax: MEC >10, > 20 and 10-20 was calculated over the entire 7 d of dosing. Maximising the percentage of animals with a Cmax: MEC of 10-20 was the criteria for “goodness” of a regimen. Supplementary pharmacokinetic parameters were also derived from the model parameters: AUCss (determined from dose and clearance), distribution (t1/2α) and terminal (t1/2β) half-life (calculated from the individual values of the pharmacokinetic parameters for each animal), Cmax (derived directly from the data as the concentration at the end of each infusion).

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Chapter 4 RESULTS

4.1 Safety

All cats completed the pharmacokinetic study and no changes were seen on complete blood counts and multiple biochemical analyses after consecutive daily dosing. Physical examination parameters were normal throughout the study except for minor transient hyperthermia in one cat (range 39.6-39.8 °C, reference range 37.5 –39.1°C) during the first caspofungin transfusion only, and mild transient diarrhoea that resolved within 24 h of the final caspofungin infusion on Day 8 in another. No adverse effects at the injection site were noted. One of the single dose cats had extravasation of its caspofungin infusion, there were no adverse effects associated with this. The caspofungin plasma concentration data was excluded for this cat.

4.2. Non-compartmental pharmacokinetic analysis

The mean and 95% CI of calculated pharmacokinetic parameters for the single and multi-dose study are presented in Table 4.1. The mean plasma drug concentration- time curve for single dose intravenous caspofungin acetate administration is presented in Figure 4.1. Caspofungin displays first-order-elimination at the dose administered, and the terminal half-life (T1/2) following a single intravenous dose is long (median 15.5 h, range: 12.8 – 18.6 h). Peak concentration (Cmax) of intravenous caspofungin for cats was recorded at 1 hour, at the time of completion of infusion (range 1 – 1.5 h) and ranged from 11.37 to 17.20 mg/L (mean of 14.15 mg/L). The volume of distribution (VD) had a mean value of 0.09 L/kg (range of 0.07 – 0.13 L/Kg) and the clearance (CL) was 4.1 mL/kg/hr (range of 2.8 – 4.9 mL/kg/hr).

The mean trough concentration after achieving steady state was 5.88 mg/L (range 3.56 – 7.78 mg/L) after 3 days of intravenous dosing. The volume of distribution (VD) had a mean value of 0.06 L/kg (range of 0.05–0.08 L/kg). Peak concentration was seen 1-1.5 h after administration (mean of 1 h) and ranged from 14.9 to 24.3 mg/L 40

(mean of 19.10 mg/L). The steady state accumulation ratio was 1.39.

Figure 4.1. Mean plasma concentration-time curve for intravenous (IV) caspofungin acetate (1 mg/kg) delivered over 1 h.

Mean plasma caspofungin concentration versus time curve 16.00 µ 14.00 12.00 10.00 8.00 6.00

g/mL) 4.00 2.00 0.00 0 5 10 15 20 25 30 Time (h) Caspofungin Plasma Concentration ( Table 4.1. Pharmacokinetic parameters following administration of a single 1 mg/kg dose of caspofungin acetate IV over 1hr and after reaching steady state after consecutive daily doses of 1 mg/kg IV.

Parameter (units) Single Dose Mean Steady State Mean (1 mg/kg, n=7)a (95% CIs) (1 mg/kg, n=6) (95% CIs)

Tmax (h) 1.1 (0.9, 1.25) 1.13 (0.9, 1.34)

Cmax (μg/mL) 14.38 (12.4, 16.35) 19.73 (15.79, 23.67)

Css,trough (μg/mL) 5.88 (4.39, 7.38)

AUC(0-∞) 251.32 (200.95, 301.69) 349.98 (233.11, 466.85)

T1/2 (h) 15.5 (13.1, 17.8) 14.5 (11.1, 17.9)

CL/F (mL/hr/kg) 4.12 (3.45, 4.78) 3.16 (2.05, 4.27)

V/F (L/kg) 0.09 (0.07, 0.11) 0.06 (0.05, 0.08)

Accumulation ratio 1.52

a Data from one cat was excluded due to extravasation of caspofungin during infusion.

Cmax, maximum concentration; AUC0-∞, area under the plasma concentration–time curve extrapolated to infinity; Css, trough,

minimal concentration prior to each daily dose; T1/2, elimination half-life; CL/F, apparent clearance; V/F, apparent volume of distribution.

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Figure 4.2. Mean plasma concentration-time curve for intravenous (IV) caspofungin acetate (1 mg/kg) after reaching steady state.

Mean plasma caspofungin concentration at steady state 20.00 g/mL)

µ 15.00

10.00

5.00 Caspofungin Concentration (

0.00 0 5 10 15 20 25 Time (h)

4.3. Pharmacokinetic modelling

The two-compartment linear model provided the best description of the plasma concentration-time profiles of caspofungin in cats compared to a 1-compartment pharmacokinetic model, the objective function value is greater than 200 (∆OBJ >200). The two-compartment base model included proportional and additive residual error terms, and random effects on clearance (CL) and the central volume of distribution (V1). Clearance and V1 were highly correlated. This was best described using a scale factor for the PPV on V1 as a function of the population parameter variability (PPV) on CL (∆OBJ >100) in order to prevent very high shrinkage on V1. Inclusion of PPV on the peripheral volume of distribution (V2) and the inter- compartmental clearance (Q) had no effect on the OBJ (∆OBJ >0.1), gave very low estimates for PPV and resulted in the failure of the covariance step. Removal of allometric scaling for weight resulted in a worsening of model fit (∆OBJ >6), and so was retained in the final model. No covariates demonstrated evidence of predictive performance, and none were included in the final model. The population pharmacokinetic parameter estimates of the final pharmacokinetic model for 42 caspofungin in cats are presented in Table 4.2.

Table 4.2. Population pharmacokinetic parameters for caspofungin in cats.

Parameter (units) Estimate %RSE

Structural

CL (mL/h) * (WT/4)0.75 17.5 7

V1 (mL) * (WT/4) 214 8.6

V2 (mL) * (WT/4) 143 8.3

Q (mL/h) * (WT/4)0.75 150 20

Between subject variability (BSV) %CV (% shrinkage)

CL 18 (0) 21

V/F BSV scale factor 1.05 (na) 8.4

Residual unexplained variability (RUV)

Proportional (%CV) 7.8 18

Additive (SD, µg/mL) 0.61 20

CL, clearance; V1, central volume of distribution; V2, peripheral volume of distribution; Q, inter-compartmental clearance; RSE, relative standard error.

All model parameters were estimated with very good precision (7-21% RSE). The inter-individual variability for clearance was low (18%), and both had minimal shrinkage (<1%). There was good agreement between the observed and predicted population as well as individually predicted concentrations, with no evidence of bias, consistent with unbiased plots of conditional weighted residuals versus concentration and time after dose. 43

The visual predictive check based on 1000 simulations of the final pharmacokinetic (PK) model suggested acceptable fit of the model to the caspofungin plasma concentration-time data. Figure 4.3 shows the visual predictive check for simulations based on initial 24 hours and final dose at day 7.

Figure 4.3. Visual predictive check plot for 1000 simulations of the final pharmacokinetic model and observed data. Solid lines represent the median and dashed lines represent the upper and lower 95% confidence intervals of the observed data (black) and prediction intervals of the final model (red). Circles represent the raw observed data.

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4.4 Caspofungin Dose Regimen Simulations

Table 4.3 summarises the caspofungin model-independent pharmacokinetic parameters and exposure indices from the “best” simulations, as well as the regimen employed in the original study (1 mg/kg every q24 h). While increasing the infusion duration from 1 h to 2 h or 4 h had some impact on the observed Cmax, this has minimal impact of the different dose regimens in the ability to achieve target exposures. As a result, only the results from 1 h caspofungin infusions are presented here. Other dose regimens and infusion durations are presented in the supplementary table accompanying appendix 1. Doses above 1 mg/kg resulted in excessively high exposures, which were less able to meet the target Cmax:MEC ratio.

Table 4.3. Summary of Caspofungin model-independent pharmacokinetic parameters and exposure indices from simulations which result in the maximum % animals 10

45

Metric Regimen Mean Median SD Minimum Maximum

Distribution t1/2 (hr) 0.388 0.387 0.0287 0.28 0.481

Terminal t1/2 (hr) 14.5 14.5 0.963 12 19.4

AUC0-τ,ss (μg.h/L)* 1mg/kg 232 230 40.7 125 436

AUC0-τ,ss (μg.h/L)* 0.75mg/kg 173 170 30.7 88.8 287

First dose

Cmax first dose (μg/mL) 1mg/kg 14.8 15 2.11 8.55 23.8

Cmax first dose (μg/mL) 0.75mg/kg 11 10.8 1.61 6.01 16.6

AUC > MIC first dose q24h 1mg/kg 133 133 22 69 227 (μg.h/L)**

AUC > MIC first dose q72h 1mg/kg 163 159 36.4 75.9 313 (μg.h/L)**

AUC > MIC first dose q24h 93.2 92.5 16.6 42.7 148 (μg.h/L)** 0.75mg/kg

Last dose

Cmax last dose (μg/mL) q24h 1mg/kg 19.8 19.6 3.23 10.9 34.2

Cmax last dose (μg/mL) q72h 1mg/kg 15.4 15.3 2.38 9.27 23.8

Cmax last dose (μg/mL) q24h 14.7 14.2 2.42 7.94 22.9 0.75mg/kg

AUC > MIC last dose q24h 1mg/kg 1350 1340 253 662 2560 (μg.h/L)***

AUC > MIC last dose q72h 1mg/kg 472 464 102 227 891 (μg.h/L)***

AUC > MIC last dose q24h 958 945 191 424 1650 (μg.h/L)*** 0.75mg/kg

* AUCss,0-Ƭ = the AUC within an inter-dosing interval (0- Ƭ), at steady-state, which is also equivalent to the AUC0-∞ if only a single dose were given. This is identical for regimens that employ the same total dose (i.e. the 1 mg/kg q24 h and 1 mg/kg q72 h). **AUC > MEC first dose: this is the AUC > MIC for the first dosing interval. For the q24 regimens this is 24 h. For the q72 h regimen this is 72 h. The AUC > MEC in the first 24 h for the q72 h 1 mg/kg regimen will be identical to that for the q24h 1mg/kg regimen as they both employ the same 1 mg/kg dose. ***AUC > MEC last dose: this is the cumulative AUC>MIC during the entire 7-day dosing period.

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In the typical 4kg cat, a 1 mg/kg daily caspofungin dose results in a Cmax of 14.8 µg/mL after the first dose, while this is 19.8 µg/mL at steady-state, with trough concentrations of caspofungin approximately 5 µg/mL. In contrast, when given every three days, a 1 mg/kg dose reaches a mean Cmax of 15.4 µg/mL and the average trough caspofungin concentrations are less than 1 µg/mL. A 0.75 mg/kg dose daily achieves a mean caspofungin Cmax of 11.0 µg/mL after the first dose and 14.7 µg/mL at steady-state, with average trough concentrations of approximately 4 µg/mL.

Plots of the median and 90% CI’s of the predicted caspofungin concentrations over time are provided in Figure 4.4.

Figure 4.4. Plots of caspofungin concentration over time.

A) 0.75 mg/kg dosing daily for 7 days, B) 1 mg/kg dosing daily for 7 days, C) 1 mg/kg every 72 hours for 7 days. Solid black line indicates the median; shaded ribbon shows the 90% CI. Dashed black lines show the putative upper and lower Cmax targets.

Figure 4.5 shows the predicted distribution of the Cmax:MEC ratio over time. The maximal percentage of animals with a Cmax:MEC ratio of 10 - 20 occurred at 0.75 mg/kg once daily (q24 h) and 1 mg/kg every 3 days (q72 h). At the peak concentration on the first day of dosing with 0.75 mg/kg, 71% of animals were within the optimal Cmax: MEC, by the third and later daily doses this reached 97%. With 1 mg/kg dosing, more than 96% of animals were within the target ratio after the first dose, and with continued q72 h dosing, this remained relatively constant for the remaining 7 days. Despite almost all animals being above the lower threshold, 47 continued daily dosing with 1 mg/kg (the regimen employed in the original trial), resulted in fewer animals within the target window after subsequent doses, reducing to 76% on the second day, and 57% after the 7th daily dose, due to 43% of animals having a Cmax:MEC ratio greater than 20.

Figure 4.5. Plots of caspofungin Cmax:MEC ratio over time.

A) 0.75 mg/kg dosing daily for 7 days, B) 1 mg/kg daily dosing for 7 days, C) 1 mg/kg every 72 hours for 7 days. Red solid line indicates the median, shaded ribbons show the 95%, 90%, 75% and 50% CI’s in decreasingly dark shading. Dashed black lines show the putative upper and lower Cmax:MEC ratio limits.

A loading dose regimen, utilising 1 mg/kg of caspofungin on day 1 followed by 0.75 mg/kg daily thereafter, resulted in 98% of cats falling within the optimal Cmax:MEC ratio from the first dose until the end of the study. Figure 4.6 shows the predicted distribution of the Cmax:MEC ratio over time for the loading dose regimen.

The differences between the regimens in terms of time-course of concentration relative to the MEC are highlighted in Table 4.3, demonstrating that the 1 mg/kg q72 h regimen results in a 50% lower AUC>MEC compared to the 0.75 mg/kg q24 h regimen.

48

Figure 4.6. Plots of caspofungin Cmax:MEC ratio over time utilising a 1mg/kg loading dose on day 1 followed by 0.75 mg/kg daily thereafter.

Red solid line indicates the median, shaded ribbons show the 95%, 90%, 75% and 50% CI's in increasingly dark shading. Dashed black lines show the putative upper and lower Cmax:MEC ratio limits.

49

Chapter 5 DISCUSSION

This study investigated the pharmacokinetics of caspofungin in healthy adult cats, finding that it is well tolerated and reaches therapeutic plasma levels at the dosing regimen studied. The over-arching goal of antifungal therapy is to achieve adequate drug concentration at the site of infection for the appropriate duration. Using a modelling and simulation approach we have also shown that doses of 0.75 mg/kg or 1 mg/kg once daily, as well as 1 mg/kg every 72 hours, will reach target therapeutic levels within serum. 98% of cats will also achieve the target therapeutic level after the first dose when a loading dose of 1 mg/kg is given on day 1 followed by 0.75 mg/kg daily. It has previously been shown that the outcomes of infection at other tissue sites correlates well with plasma concentrations of the drug [279].

The intravenous caspofungin concentration-time data in cats were best described by a linear population pharmacokinetic model which was in agreement with data collected from other species including rodents (mice, rats), lagomorphs (rabbits) and primates (chimpanzees, rhesus monkeys and humans) [213, 280]. It reaches maximal serum concentration levels at the end of the intravenous infusion. This was further supported by the population model. As expected, the elimination half-life of caspofungin in cats (14.5 ± 0.96 h) was longer than that in healthy human adults administered an equivalent 70 mg dose (9.29 ± 1.96 h) [212]. Steady state was achieved at approximately 72 h with trough concentrations of caspofungin in cats at 1 mg/kg and 0.75 mg/kg daily of approximately 5 µg/mL and 4 µg/mL respectively. In contrast, humans given 70 mg/kg daily reportedly only achieved steady state during the third week of administration with a lower trough concentration of 2.57 ± 0.34 µg/mL, as a result of a much longer terminal half-life in humans (40 – 50 h) compared to cats (15 h) [212]. The clearance reported in this study, in the typical 4kg cat (18 mL/min) is larger on a per kg basis than that reported in the typical 70 kg human (10 mL/min). As Stone, Holland (212) did not report the volume of distribution 50 a direct comparison cannot be made, however it would appear that humans likely have a greater volume of distribution per kg compared to cats.

Studies conducted in humans and rats have shown that following initial distribution caspofungin is taken up by the liver and slowly metabolised; primarily in the liver, as well as the adrenal glands and spleen [210]. Furthermore, metabolism of caspofungin to inactive metabolites occurs by non-enzymatic hydrolysis and N- acetylation [45, 210, 214]. The OATP-1B1 transporter pathway is responsible for hepatic uptake of caspofungin in rats, as well as the transport of bile which eliminates the degradation products of caspofungin [210, 215]. Cats are deficient in NAT2 and possess a single NAT similar to human NAT1 but with lower affinity and activity; therefore, it is likely that other pathways are involved in its metabolism and this warrants further investigation [42, 49]. Metabolic deficiencies in cats have been shown to occur mostly in phase II metabolism, such as with glucuronidation, acetylation, and sulfation reactions. Hence a proposed mechanism of feline caspofungin metabolism may be through enhanced phase I metabolism via hydrolysis or other mechanisms [42].

Caspofungin exerts its antifungal activity on the b-glucans, i.e. glucose polymers that are a major cell wall structural component in fungi, plants and some bacteria, but not in mammals. Hence adverse effects in mammalian species are expected to be low [281]. In this study, caspofungin was well tolerated by cats with only two adverse events observed – transient fever and diarrhoea. Adverse events in healthy humans are minimal and are mostly due to infusion reactions such as fever, gastrointestinal signs, phlebitis and headache [1, 41, 214]. Histamine – mediated symptoms; including anaphylaxis, rashes, facial swelling, pruritus, bronchospasm and cardiac arrhythmias; have rarely been reported [208, 242, 282]. Manifestations of acute anaphylaxis are species-dependent. Thus, animal-models of disease need to account for such species differences and prospective studies of the effect of caspofungin in cats with active fungal infections are warranted.

51

Liver enzyme elevations did not occur in any cats in this study and were uncommon in healthy caspofungin-treated humans [212]. However, adverse events have been shown to be higher in patients with invasive fungal infections, co-morbidities and altered drug metabolism. For example, rabbits in a model of invasive aspergillosis had significantly slower clearance of anidulafungin associated with a higher plasma concentration of drug at the end of the dosing interval compared to healthy individuals [283]. Hepatotoxicity, manifesting as elevated liver enzymes (ALT, AST or ALP), and infusion reactions are common in human patients with invasive fungal infections treated with caspofungin [284]. The most common clinical adverse events were fever (7.5%), chills (5.3%), nausea or vomiting (5.3%) and phlebitis (3.5%) [284].

Caspofungin is also registered for use in patients that are unable to tolerate other antifungals due to hepatic or renal insufficiency, as well as for empirical therapy for presumed fungal infections in febrile neutropenic patients [41, 116]. Studies in humans and rodents have shown that caspofungin is not metabolised or excreted via the renal system [85, 212, 213]. It may, therefore, be a good option in elderly cats with renal insufficiency, or cats experiencing adverse effects from amphotericin B or azole therapy, however the route of metabolism and excretion requires further evaluation in cats. Poor clearance of antifungal drugs in sick patients may also affect patient outcome and predispose to the development of paradoxical growth or trailing effect at drug concentrations greater than the MIC/MEC, leading to increased growth of fungus and negative patient outcomes [199, 285, 286].

The fungi responsible for feline sino-orbital aspergillosis are from the Aspergillus viridinutans complex, within Aspergillus section Fumigati [287]. A. felis, a cryptic species of A. virdinutans, is most commonly implicated as the causative agent, and has also been associated with invasive aspergillosis in humans and dogs [31]. Antifungal susceptibility was tested for thirteen A. felis isolates from cats by Barrs, van Doorn (31), caspofungin displayed a MEC of less than 0.06 µg/mL in all but one isolate which was 2 µg/mL. The majority of isolates had MICs greater than 0.06 µg/mL for both amphotericin B and the azoles tested; for voriconazole MICs were 52 greater than 1 µg/mL in 9/13 isolates [31]. In the current study, at a dose of 1 mg/kg q24 h Cmax and Cmin were well in excess of the highest MECs reported for Aspergillus viridinutans complex species. However, other cryptic species in section Fumigati reported to cause invasive aspergillosis in humans and cats have shown decreased susceptibility to caspofungin, including some pathogenic isolates of A. lentulus from human cases which had MECs of caspofungin of 4 - >32 µg/mL [34].

The trough plasma caspofungin concentration in this study was no less than 2.78 µg/mL for all animals for the duration of the study period, which is above the MEC reported for Aspergillus Section Fumigati isolates, as well as greater than the recommended plasma concentration (1 µg/mL) in humans [34, 35, 221, 288]. In the pharmacokinetic model utilising this dose, 95% of the population achieved a trough concentration of approximately 3.75 μg/L, with the average around 5 μg/L, which is well above the MEC and recommended plasma concentrations. As previously mentioned, paradoxical growth effect can lead to negative patient outcomes. Thus, it is important to utilise a dosing regimen which does not greatly exceed the MEC for the isolate and to perform appropriate susceptibility testing and therapeutic monitoring.

Appropriate plasma concentration of caspofungin is a potential predictor of efficacy, as it has been shown to have concentration-dependent activity against Aspergillus spp., unlike the azoles which are time-dependent [231]. Utilising the Wiederhold, Kontoyiannis (231) target for therapeutic doses of caspofungin, the simulated data shows that an optimal Cmax: MEC of 10-20 is achieved using 0.75 mg/kg q24 h or 1 mg/kg q3 days. In the regimen initially studied (1 mg/kg q24 h), and extrapolated from the human dosage, the Cmax:MEC ratio is too high in a significant number of cats (42.6% greater than 20 after 7 consecutive doses). At this dose with a 3-day inter-dosing interval, Cmin was shown to be well below 1 μg/mL in more than 95% of the population as the prolonged dosing interval does not allow for drug accumulation.

Using a daily dose of 0.75 mg/kg, the Cmax: MEC is below 20 and Cmin is greater than 2.5 μg/mL for more than 95% of the population. This difference in exposure between 53 doses is also highlighted by the 50% reduction in the cumulative AUC above the MEC for the 1 mg/kg q72h regimen compared to 0.75 mg/kg q24h. One week of treatment was considered a long enough time to ensure plasma concentrations were at steady-state. Measuring the plasma concentrations at steady state is clinically relevant as fungal infections typically require long-term treatment to achieve resolution [41]. Although both 1 mg/kg q72h and 0.75 mg/kg q24h provide very similar results for Cmax: MEC, the former regimen in vastly superior in regard to AUC

> MEC. The Cmax:MEC ratio appears promising utilising a dosage of 1 mg/kg every 72 h, potentially to improve treatment compliance; however this regimen does not appear appropriate when considering trough plasma concentration and drug accumulation to achieve steady state. Therefore based on the modelling data, a daily dose of 0.75 mg/kg is most likely to meet the ideal Cmax:MEC, and provide consistent drug exposure between doses, thus improving patient outcomes. Alternatively, utilising a loading dose regimen of 1 mg/kg on day 1, followed by 0.75 mg/kg daily will rapidly achieve steady state, as well as provide continual exposure between dosing.

The use of pharmacokinetic modelling aimed to reduce the bias associated with the small number of individuals in the original pharmacokinetic study. The visual predictive check shows that the pharmacokinetic data presented here is a good representation of the population. The two-stage method was utilised for the pharmacokinetic study in order to minimise bias associated with pharmacokinetic calculations, such as the naïve pooled data method, which do not take into account inter- and intra- individual variability. However, as the data was estimated from each individual concentration versus time curve some upward bias could not be avoided. The two-stage approach is resource intensive and requires repeated studies in different populations, for example feline breed differences have not been taken into account in our study design and thus a pharmacokinetic model is a superior method of assessing the drugs utility in a healthy cat population and allows for a more accurate dosing regimen to be achieved.

54

The relationship between antifungal in vivo susceptibility, dosages, drug concentrations within various body compartments and toxicological effects are not completely understood. Although our pharmacokinetic analyses have attempted to predict a regimen appropriate for safe and effective use of caspofungin using in vitro MEC values, inherent biological factors can affect therapeutic outcome. For example, in SOA systemic therapy may not be sufficient to penetrate the site of infection and reduce fungal burden. In addition, this work is based upon total plasma concentrations, and not unbound drug, which may further confound the results as caspofungin is highly protein bound (>95%) [85, 210]. The plasma free fraction in the cat is unknown. Nevertheless, the current clinical regimen provides total plasma concentrations consistent with the Cmax:MEC ratio proposed in the present analyses, suggesting that the simulations are likely to be clinically relevant. Further pharmacodynamic studies are, therefore, required which can be linked with the pharmacokinetic data presented here, prior to a robust prospective clinical study to document clinical efficacy and safety of the proposed regimens in feline patients affected by sino-orbital aspergillosis.

55

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Appendix I

RESEARCH ARTICLE Pharmacokinetics of caspofungin acetate to guide optimal dosing in cats

Jana Leshinsky1, Andrew McLachlan2,3, David J. R. Foster4, Ross Norris5,6,7, Vanessa R. Barrs1*

1 Sydney School of Veterinary Science, Faculty of Science, The University of Sydney, Camperdown, New South Wales, Australia, 2 Faculty of Pharmacy and Education and Research on Ageing, The University of Sydney, Camperdown, New South Wales, Australia, 3 Concord Hospital, Concord, New South Wales, Australia, 4 School of Pharmacy and Medical Sciences, Australian Centre for Pharmacometrics, Sansom Institute for Health Research, University of South Australia, Adelaide, South Australia, Australia, 5 Clinical Pharmacology Division, SydPath, St Vincent's Hospital, Darlinghurst, New South Wales, Australia, 6 Clinical a1111111111 School, University of New South Wales, Darlinghurst, New South Wales, Australia, 7 School of Pharmacy, Griffith University, Gold Coast, Queensland, Australia a1111111111 a1111111111 * [email protected] a1111111111 a1111111111 Abstract

Cats are the most common mammal to develop invasive fungal rhinosinusitis caused by

OPEN ACCESS cryptic species in Aspergillus section Fumigati that are resistant to azoles but susceptible to caspofungin. In this study nonlinear mixed-effects pharmacokinetic modeling and simulation Citation: Leshinsky J, McLachlan A, Foster DJR, Norris R, Barrs VR (2017) Pharmacokinetics of was used to investigate caspofungin pharmacokinetics and explore dosing regimens in cats caspofungin acetate to guide optimal dosing in using caspofungin minimum effective concentrations (MECs). Plasma concentrations in cats. PLoS ONE 12(6): e0178783. https://doi.org/ healthy cats were determined using HPLC-MS/MS after administration of a single and 10.1371/journal.pone.0178783 seven consecutive daily intravenous doses of 1 mg/kg caspofungin. In the final pharmacoki- Editor: Douglas Thamm, Colorado State University, netic model an optimum maximum concentration (Cmax): MEC ratio of 10±20 was used to UNITED STATES guide caspofungin efficacy. Simulations were performed for dosing regimens (doses 0.25±2 Received: March 7, 2017 mg/kg and 6±72 h dosing intervals) with and without inclusion of a loading dose. Using a 1

Accepted: May 18, 2017 mg/kg dose Cmax first dose was 14.8 μg/mL, Cmax at steady state was 19.8 μg/mL, Cmin was

Published: June 2, 2017 5 μg/mL and Cmax: MEC was >20 in 42.6% of cats after multiple doses. An optimal Cmax: MEC ratio was achieved in caspofungin simulations using 0.75 mg/kg q 24 h or 1 mg/kg q Copyright: © 2017 Leshinsky et al. This is an open access article distributed under the terms of the 72h. However, at 1 mg/kg q 72h, Cmin was < MEC (<1 μg/mL) in over 95% of the population. Creative Commons Attribution License, which Using a loading dose of 1 mg/kg and a daily dose of 0.75 mg/kg thereafter, the Cmax: MEC permits unrestricted use, distribution, and was optimal and Cmin was > 2.5 μg/mL for 98% of the population. Based on the modeling reproduction in any medium, provided the original data this dosing regimen is likely to achieve target therapeutic concentrations, meet the pro- author and source are credited. posed Cmax: MEC window and provide consistent exposure between doses. Data Availability Statement: All relevant data are within the paper and its Supporting Information files.

Funding: This study was funded by the Australian Companion Animal Health Foundation 009/2015. The funder had no role in study design, data collection and analysis, decision to publish, or Introduction preparation of the manuscript. Cats are the most common mammal to develop naturally occurring invasive fungal rhinosinu- Competing interests: The authors have declared sitis caused by cryptic species of Aspergillus in section Fumigati [1]. Sino-orbital aspergillosis that no competing interests exist. occurs in immunocompetent cats, is usually fatal and is most frequently caused by two

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emerging pathogens in the Aspergillus viridinutans species complex, A. felis and A. udagawae [1]. These cryptic species in section Fumigati also cause invasive fungal infection (IFIs) in humans, usually in the form of invasive pulmonary aspergillosis [1–3]. In both humans and cats A. udagawae and A. felis cause infections with a protracted clinical course that spread across anatomical planes to involve contiguous tissues and are refractory to standard therapy [2, 4]. The minimum inhibitory concentrations (MICs) of azole and polyene antifungal agents against Aspergillus viridinutans complex species are higher than those for A. fumigatus [3]. Caspofungin is an approved echinocandin for treatment of human patients with invasive aspergillosis refractory to other antifungal therapy [5–12] and was used to cure a case of feline sino-orbital aspergillosis caused by A. felis that failed treatment with azoles and amphotericin B [4]. The pharmacokinetics of caspofungin have been evaluated in rodents, lagomorphs and primates [13, 14]. Among companion animals, the pharmacokinetics of mica- fungin, but not caspofungin have been determined in dogs and no echinocandins have been evaluated in felines [15]. The pharmacokinetics of caspofungin in cats cannot be extrapolated from other mammals studied thus far since cats have substantial differences in drug metabolism compared to other mammals, including deficiencies in multiple phase II metabolic pathways including N-acetyl- transferase (NAT) [16]. Our objective was to determine the pharmacokinetics of caspofungin in healthy cats after an intravenous dose of 1 mg/kg and after 7 consecutive daily doses. This study used a modeling and simulation approach to explore caspofungin dosing regimens in

cats designed to examine Cmax: MEC ratios after intravenous caspofungin infusions.

Materials and methods Pharmacokinetic study Animals. Eight healthy adult desexed domestic shorthair cats (4 females, 4 males) with mean age and body weight of 3.94 ± 1.84 years and 4.4 ± 0.56 kg, respectively were recruited for the study. The cats were healthy cats from a research colony owned by Eurofins SCEC Pty Ltd. (www.scec.net.au) and were returned at the completion of the study in good health. Results of a complete blood count, serum biochemical analyses and urine specific gravity per- formed 5 days prior to the start of the study were within reference intervals for all cats. Cats were housed individually and acclimatized to the clinical environment for three days prior to the start of the study with free access to food and water. The study was approved by the Animal Ethics Committee (AEC) of The University of Sydney (Approval no. 2015/775, 6th March, 2015). Drug administration and blood sampling. For blood sampling, a 20-guage 8 cm triple- lumen central venous catheter (MILACATH1, MILA International, Inc., Kentucky, USA) was inserted into the jugular vein using a modified Seldinger technique after sedation with IV medetomidine (10 μg/kg; Domitor1, Pfizer Animal Health Australia, West Ryde, NSW) and butorphanol (0.1 mg/kg; Ilium Butorgesic1, Troy Laboratories Pty Limited, Glendenning, NSW), 24 h prior to starting the study. For drug administration, a peripheral catheter (22G IV radiopaque catheter; OPTIVA1, Smiths Medical International Ltd, Lanchashire, UK) was placed into a cephalic vein. For the single dose study (n = 8 cats) caspofungin acetate (Canci- das1, Merck Sharp & Dohme (Australia) PTY Limited, South Granville, NSW) was adminis- tered by IV infusion (1 mg/kg), 0.2 mg/mL in sterile saline solution (0.9% sodium chloride, Abbott Laboratories, Abbott Park, IL) over 1 h. For the multiple consecutive daily dosing study (n = 6 cats, 3 male, 3 female) administration of caspofungin acetate (1 mg/kg IV infusion over 1 h) was repeated once daily (at the same time each day) for a further 6 days.

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Blood (1 mL) was collected in lithium-heparin immediately before drug administration (time 0), and after IV administration at 0.5, 0.75, 1, 1.25, 1.5, 2, 3, 6, 9, 12, and 24 h. Blood sam- ples were immediately centrifuged (12, 000 g for 5 min) and plasma was stored at -80˚C until assay. For the multi-dose study, a single blood sample was collected before each daily infusion (48, 72, 96 and 120 h after the first dose) to ascertain steady state plasma concentrations and blood sampling was repeated at the same times as day 1 on day 7. Repeat biochemical analyses were performed on Day 8, 24 h after the final caspofungin dose and pharmacokinetic blood sample collection. Analytical method. Caspofungin concentrations in cat plasma were determined using HPLC-MS/MS (tandem mass spectrometry). Blood samples were prepared for assay using protein precipitation by adding methanol (80 μL) to 20 μL of calibrators, controls (drug free) and unknown samples using a stable isotope of caspofungin as internal standard (in methanol). Analytical reference standard for caspofungin was supplied by Merck Sharp & Dohme (Whitehouse Station, New Jersey 08889, USA). Chromatography and analysis was performed on a Shimadzu UPLC coupled to an 8050 tandem mass spectrometer in positive ion ESI mode, monitoring the transition 547.6 > 538.5 and internal standard transition

549.5 > 86.2. A Waters BEH C18 column was used with gradient elution with mobile phase A (water with 0.1% formic acid) and mobile Phase B (acetonitrile containing 0.1% formic acid). The mobile phase gradient involved 100% A for 0.5 minutes and a linear gradient to 0% A by 4.5 minutes. After raising the gradient to 100% B at 4.5 minutes it was held there for 0.5 minutes before returning to initial conditions for a further minute, making a total run time of 6 minutes. The assay was validated in accordance with the US FDA Guidelines for industry for bioana- lytical method validation utilising pooled plasma from six untreated cats with known concen- trations of caspofungin reference standard. Linearity was demonstrated over the caspofungin concentration range of 0.1 to 10.0 μg/mL. The assay accuracy and precision (4 replicates on 3 occasions of spiked caspofungin plasma samples at 0.2 and 9.0 μg/mL) showed total precision of 12.0 and 4.3% (expressed as co-efficient of variation), respectively. Accuracy was 103.4 and 96.7%, respectively. Carry-over was demonstrated to be no more than 1.0% of the lowest stan- dard in blank matrix following injection of the highest standard (10.0 μg/mL).

Population pharmacokinetics analysis and dose regimen simulations Methods software. Pharmacokinetic model development employed nonlinear mixed- effects modeling using NONMEM version 7.3 [17], with the Wings for NONMEM 7.3interface (http://wfn.sourceforge.net) and IFort compiler. Modeling was performed using a Dell Power- Edge R910 server with 4 by 10 core Xeon 2.26-Ghz processors running Windows Server 2008 R2 Enterprise 64- bit software. Data manipulation and post-run processing of NONMEM output was conducted using the R data analysis language (Version 3.2.2) and the packages ggplot2, GGally, foreign, tidyr, Hmisc, gdata, doBy, plyr, grid, stringr with associated depen- dencies [17–28]. General modeling strategy. The base pharmacokinetic model was developed in a step- wise manner. Pharmacokinetic models were coded using the built-in ADVAN subroutines of NONMEM. Linear kinetic models with 1 and 2 compartments were evaluated. The First Order Conditional Estimation (FOCE) method was used to fit models. The base model was selected on the basis of mechanistic plausibility, visual inspection of goodness-of-fit diagnostic plots, the precision of parameter estimates (se% <30% for fixed, <50% for random effects parameters), and the lowest value of the Akaike’s information criterion (AIC; Eq 1) in accor- dance with the number of parameters and the final NONMEM derived objective function

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value (OBJ). The base model was also required to pass the covariance step.

AIC ¼ OBJ þ 2 Ã number of parameters ð1Þ

Unless stated otherwise, population parameter variability (PPV) was represented using an exponential error model (Eq 2):

Zj Pj ¼ TVP Ã e ð2Þ

th where Pj is the individual value for the parameter in the j individual, TVP is the typical popu- lation value of P and ηj is an independent random variable with a mean of zero and variance ω2. PPV was systematically examined on each fixed-effect parameter. Models with and without covariance for P were investigated using the OMEGA BLOCK functionality of NONMEM.

A combined proportional (θprop) and additive (θadd) residual unexplained error model of the caspofungin concentrations (C) was used, where estimation of a THETA was employed and epsilon was fixed to zero (Eq 3): qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ^ 2 ^ 2 2 Cij ¼ Cij þ yprop à Cij þ yadd ð3Þ

th th ^ Cij is the i concentration measured in the j individual, Cij is the model predicted Cij, and

θprop and θadd are parameters representing the proportional and additive residual error, respec- tively. The influence of the additive component of the error was also examined by a compari- son with a proportional only error model. All base models were investigated with allometric scaling for total body weight (TBW) ref- erenced to 4 kg and an exponent fixed of 0.75 for clearance parameters and 1 for volumes, although during base model development models was explored without allometric scaling on any parameter. Covariate model building. The base model was screened for the influence of cat sex, guided by plots of individual parameter random effects versus sex if shrinkage was low. Sex was only retained in the model if there was a significant improvement in OBJ at the p <0.01 level. Model evaluation. Visual Predictive Checks (VPC) were used to assess the appropriate- ness of the candidate base and final models, faceted for any included covariates as appropriate. For the VPC’s, the median, 5th and 95th percentiles of the prediction-corrected observations were compared against the empirical 95% confidence intervals (CI) of the median, 5th and 95th percentiles of caspofungin concentrations from 1000 simulations of the index (original) data- set. The predictive performance of the model was considered acceptable if the median, 5th and 95th percentiles of the prediction-corrected observed and simulated data agreed well. Caspofungin dose regimen simulations. The final pharmacokinetic model for caspofun-

gin in cats was used to perform dosing simulations designed to examine Cmax: minimum effec- tive concentration (MEC) ratios following intravenous infusions of caspofungin. Simulations were performed utilising total drug in serum rather than unbound drug, as published data available for MEC values have only been reported for the total drug. Based on data from a

murine model of invasive pulmonary aspergillosis an optimum Cmax: MEC ratio of 10–20 was chosen as a marker of caspofungin dose efficacy [29]. Simulations were performed for dosing regimens of 0.25 mg/kg at 6 h intervals; 0.5 mg/kg at 6 h, 12 h and 24 h dosing intervals; 0.75 mg/kg and 1 mg/kg at 24 h, 48 h and 72 h intervals; 2 mg/kg at 48 h and 72 h dosing intervals. Loading dose regimen simulations were also considered based upon the results of the con- stant-dose regimens. A MEC value of 1 μg/mL was chosen based on the epidemiological cut- off value (ECV) of caspofungin for wild type A. fumigatus sens. str [30] and on antifungal

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susceptibility MEC data from clinical human and feline isolates of A. felis [1] and A. udagawae [2, 31, 32]. The duration of infusion was adjusted to 1, 2 and 4 h in the 1 mg/kg and 2 mg/kg simulations. Concentrations were simulated at 0.1 h intervals for 1000 standard 4 kg cats for

each dose regimen for a total of 7 days of dosing. The percentage of animals with a Cmax: MEC > 10, > 20 and 10–20 was calculated over the entire 7 days of dosing. Maximising the

percentage of animals with a Cmax: MEC of 10–20 was the criteria for “goodness” of a regimen. Supplementary pharmacokinetic parameters were also derived from the model parameters:

AUCss (determined from dose and clearance), distribution (t1/2α) and terminal (t1/2β) half-life (calculated from the individual values of the pharmacokinetic parameters for each animal),

Cmax (derived directly from the data as the concentration at the end of each infusion).

Results All cats completed the pharmacokinetic study and no changes were seen on complete blood counts and multiple biochemical analyses after consecutive daily dosing. Physical examination parameters were normal throughout the study except for minor transient hyperthermia in one cat (range 39.6–39.8˚C, reference range 37.5–39.1˚C) during the first caspofungin transfusion only and mild transient diarrhoea that resolved within 24 h of the final caspofungin infusion on Day 8 in another.

Pharmacokinetic modeling The two-compartment linear model provided the best (ΔOBJ >200) description of the plasma concentration-time profiles of caspofungin in cats compared to a 1-compartment pharmacoki- netic model. The two-compartment base model included both proportional and additive resid- ual error terms, and random effects on clearance (CL) and the central volume of distribution (V1). Clearance and V1 were highly correlated. This was best described using a scale factor for the PPV on V1 as a function of the PPV on CL (ΔOBJ > 100) in order to prevent very high shrinkage on V1. Inclusion of PPV on the peripheral volume of distribution (V2) and the inter-compartmental clearance (Q) had no effect on the OBJ (ΔOBJ > 0.1), gave very low esti- mates for PPV and resulted in failure of the covariance step. Removal of allometric scaling for weight resulted in a worsening of model fit (ΔOBJ > 6), and so was retained in the final model. No covariates demonstrated evidence of predictive performance, and none were included in the final model. The population pharmacokinetic parameter estimates of the final pharmacoki- netic model for caspofungin in cats are presented in Table 1. All model parameters were estimated with very good precision (7–21% RSE). The inter- individual variability for clearance was low (18%) and had minimal shrinkage (< 1%). There was good agreement between the observed and predicted population as well as individually predicted concentrations, with no evidence of bias, consistent with unbiased plots of condi- tional weighted residuals versus concentration and time after dose. The visual predictive check based on 1000 simulations of the final PK model suggested acceptable fit of the model to the caspofungin plasma concentration-time data. Fig 1 shows the visual predictive check for simulations based on initial 24 h and final dose on day 7. Caspofungin dose regimen simulations. Table 2 summarises the caspofungin model- independent pharmacokinetic parameters and exposure indices from the “best” simulations, as well as the regimen employed in the original study (1 mg/kg every q24 h). While increasing

the infusion duration from 1 h to 2 h or 4 h had some impact on the observed Cmax, this has minimal impact of the different dose regimens in the ability to achieve target exposures. As a result, only the results from 1 h caspofungin infusions are presented here. Data for other dose regimens and infusion durations are presented in supporting material (S1 Table). Similarly,

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Table 1. Population pharmacokinetic parameters for caspofungin in cats. Parameter (units) Estimate %RSE Structural CL (mL/h) * (WT/4)0.75 17.5 7 V1 (mL) * (WT/4) 214 8.6 V2 (mL) * (WT/4) 143 8.3 Q (mL/h) * (WT/4)0.75 150 20 Between subject variability (BSV) %CV (% shrinkage) CL 18 (0) 21 V/F BSV scale factor 1.05 (na) 8.4 Residual unexplained variability (RUV) Proportional (%CV) 7.8 18 Additive (SD, μg/mL) 0.61 20

CL, clearance; V1, central volume of distribution; V2, peripheral volume of distribution; Q, inter- compartmental clearance; RSE, relative standard error.

https://doi.org/10.1371/journal.pone.0178783.t001

doses above 1mg/kg resulted in excessively high exposures, which were less able to meet the

target Cmax:MEC ratio, and are not presented here. Data for other dose regimens and infusion durations are presented in supporting material (S1 Table).

In the typical 4 kg cat, a 1 mg/kg daily caspofungin dose results in a Cmax of 14.8 μg/mL on the first dose while this is 19.8 μg/mL at steady-state, with trough concentrations of caspofungin approximately 5 μg/mL. In contrast, when given every 3 days, a 1 mg/kg dose reaches a mean

Fig 1. Visual predictive check plot for 1000 simulations of the final pharmacokinetic model and observed data. Solid lines are the median and dashed lines represent the upper and lower confidence intervals of the observed data (black) and prediction intervals of the final model (red). Circles are the raw observed data. https://doi.org/10.1371/journal.pone.0178783.g001

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Table 2. Summary of caspofungin model-independent pharmacokinetic parameters and exposure indices from simulations which result in the maximum % animals 10 < Cmax/MEC < 20 ratio for three dosing regimens administered as 1 h duration infusions (0.75 mg/kg q 24 h, 1 mg/kg q 24 h and 1 mg/kg q72 h). Metric Regimen Mean Median SD Minimum Maximum

Distribution t1/2 (hr) 0.388 0.387 0.0287 0.280 0.481

Terminal t1/2 (hr) 14.5 14.5 0.963 12.0 19.4

AUCss,0-T (μg.h/mL)* 1 mg/kg 232 230 40.7 125 436

AUCss,0-T (μg.h/mL) 0.75 mg/kg 173 170 30.7 88.8 287 First dose

Cmax ®rst dose (μg/mL) 1 mg/kg 14.8 15.0 2.11 8.55 23.8

Cmax ®rst dose (μg/mL) 0.75 mg/kg 11.0 10.8 1.61 6.01 16.6 AUC > MEC ®rst dose (μg.h/mL)** 1 mg/kg q 24 h 133 133 22.0 69.0 227 AUC > MEC ®rst dose (μg.h/mL)** 1mg/kg q72 h 163 159 36.4 75.9 313 AUC > MEC ®rst dose (μg.h/mL)** 0.75mg/kg q 2 4h 93.2 92.5 16.6 42.7 148 Last dose

Cmax last dose (μg/mL) 1 mg/kg q 24 h 19.8 19.6 3.23 10.9 34.2

Cmax last dose (μg/mL) 1 mg/kg q 72 h 15.4 15.3 2.38 9.27 23.8

Cmax last dose (μg/mL) 0.75 mg/kg q24 h 14.7 14.2 2.42 7.94 22.9 AUC > MEC last dose (μg.h/mL)*** 1 mg/kg q24 h 1350 1340 253 662 2560 AUC > MEC last dose (μg.h/mL)*** 1 mg/kg q72 h 472 464 102 227 891 AUC > MEC last dose (μg.h/mL)*** 0.75mg/kg q24 h 958 945 191 424 1650

* AUCss,0-T = the AUC within an inter-dosing interval (0-T), at steady-state, which is also equivalent to the AUC0-in®nity if only a single dose were given. This is identical for regimens that employ the same total dose (ie. the 1mg/kg q24h and 1mg/kg q72h). **AUC > MEC ®rst dose: this is the AUC > MIC for the ®rst dosing interval. For the q24 regimens this is 24h. For the Q72h regimen this is 72h. The AUC > MEC in the ®rst 24h for the q72h 1mg/kg regimen will be identical to that for the q24h 1mg/kg regimen as they both employ the same 1mg/kg dose. ***AUC > MEC last dose: this is the cumulative AUC>MIC during the entire 7 day dosing period.

https://doi.org/10.1371/journal.pone.0178783.t002

Cmax of 15.4 μg/mL and the average trough caspofungin concentrations are less than 1 μg/mL. A 0.75 mg/kg dose daily achieves a mean caspofungin Cmax of 11.0 μg/mL after the first dose, 14.7 μg/mL at steady-state, with average trough concentrations of approximately 4 μg/mL. Plots of the median and 90% CI’s of the predicted caspofungin concentrations over time are

provided in Fig 2, while Fig 3 shows the predicted distribution of the Cmax:MEC ratio over time. The maximal percentage of animals with a Cmax:MEC ratio of 10–20 occurred at 0.75 mg/kg once daily (q24 h) and 1 mg/kg every 3 days (q72 h). At the peak concentration on the

first day of dosing with 0.75 mg/kg, 71% of animals were within the optimal Cmax: MEC, by the third and later daily doses this reached 97%. With 1 mg/kg dosing, > 96% of animals were within the target ratio after the first dose, and with continued q72 h dosing, this remained rela- tively constant for the remaining 7 days. Despite almost all animals being above the lower threshold, continued daily dosing with 1 mg/kg (the regimen employed in the original trial), resulted in fewer animals within the target window after subsequent doses, reducing to 76% on th the second day, and 57% after the 7 daily dose due to 43% of animals having a Cmax:MEC ratio greater than 20. A loading dose regimen, utilising 1mg/kg of caspofungin on day 1 fol-

lowed by 0.75mg/kg q 24 h thereafter, resulted in 98% of cats falling within the optimal Cmax: MEC ratio from the first dose until the end of the study. Fig 4 shows the predicted distribution

of the Cmax:MEC ratio over time for the loading dose regimen. The differences between the regimens in terms of time-course of concentration relative to the MEC are highlighted in Table 2, demonstrating that the 1 mg/kg q72 h regimen results in a 50% lower AUC > MEC compared to the 0.75 mg/kg q24 h regimen.

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Fig 2. Plots of caspofungin concentration over time. A) 0.75mg/kg dosing daily for 7 days, B) 1mg/kg dosing daily for 7 days, C) 1mg/kg every 72 hours for 7 days. Solid black line indicates the median, shaded ribbon shows the 90% CI. Dashed black lines show the putative upper and lower Cmax targets. https://doi.org/10.1371/journal.pone.0178783.g002

Discussion This study has investigated the pharmacokinetics of caspofungin in healthy adult cats, finding that it is well tolerated and reaches therapeutic plasma levels at the dosing regimen studied. Using an innovative modeling and simulation approach we have shown that doses of 0.75 mg/ kg or 1 mg/kg once daily, as well as 1 mg/kg every 72 hours, will reach target therapeutic levels. We have also shown that a loading dose of 1 mg/kg followed by 0.75mg/kg daily will enable 98% of cats to achieve the target therapeutic level after the first-dose. Caspofungin exhibits linear pharmacokinetics in cats after intravenous administration of a single 1 mg/kg dose as demonstrated in other mammals including rodents (mice, rats), lago- morphs (rabbits) and primates (chimpanzees, rhesus monkeys and humans) [13, 14]. In humans, hepatic degradation of caspofungin to inactive metabolites occurs by non-enzymatic

Fig 3. Plots of caspofungin Cmax:MEC ratio over time. A) 0.75mg/kg dosing daily for 7 days, B) 1mg/kg daily dosing for 7 days, C) 1mg/kg every 72 hours for 7 days. Red solid line indicates the median, shaded ribbons show the 95%, 90%, 75% and 50% CI's in increasingly dark shading. Dashed black lines show the putative upper and lower Cmax:MEC ratio limits. https://doi.org/10.1371/journal.pone.0178783.g003

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Fig 4. Plots of caspofungin Cmax:MEC ratio over time utilising a 1mg/kg loading dose on day 1 followed by 0.75 mg/kg daily thereafter. Red solid line indicates the median, shaded ribbons show the 95%, 90%, 75% and 50% CI's in increasingly dark shading. Dashed black lines show the putative upper and lower Cmax:MEC ratio limits. https://doi.org/10.1371/journal.pone.0178783.g004

hydrolysis and N-acetylation [33–35]. Cats are deficient in NAT2 and possess a single NAT similar to human NAT1 but with lower affinity and activity [16]. Whether this is the major pathway of caspofungin metabolism in the cat is not known. The elimination half-life of caspo- fungin in cats (14.5 ± 0.96 h) was longer than that in healthy human adults administered an equivalent 70 mg dose (9.29 ± 1.96 h) [35]. Steady state was achieved at approximately 72 h with trough concentrations of caspofungin in cats at 1 mg/kg and 0.75 mg/kg daily of approximately 5 μg/mL and 4 μg/mL, respectively. In contrast, humans given 70 mg/kg of caspofungin daily reportedly only achieved steady state during the third week of administration with a lower trough concentration of 2.57 ± 0.34 μg/ mL, as a result of a much longer terminal half-life in humans (40–50 h) compared to cats (15 h) [36]. The clearance we report in the typical 4 kg cat (18 mL/min) is larger on a per kg basis than that reported in the typical 70 kg human (10mL/min). We cannot comment on a compar- ison of volume of distribution as Stone et al [36] did not report this parameter, although it would appear that humans most likely have a larger volume of distribution per kg compared to cats. Caspofungin was well tolerated by cats with only two adverse events (AE) observed—tran- sient fever and diarrhoea. Since caspofungin exerts its fungicidal activity on the β-glucans, which are essential glucose polymer cell wall components of fungi but not mammals, AE in mammalian species are expected to be low compared to other antifungals [37]. AE in healthy humans administered caspofungin are minimal and mostly comprise infusion reactions [36]. Liver enzyme elevations did not occur in any cats in this study and were uncommon in healthy caspofungin-treated humans [36]. However, AE are higher in patients because of co-morbidi- ties and altered drug metabolism. Rabbits in an IFI model had significantly slower clearance of

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anidulafungin associated with a higher plasma concentration of drug at the end of the dosing interval compared to healthy individuals [38]. For human patients with IFIs treated with cas- pofungin, hepatotoxicity and infusion reactions are most common AEs [39]. In caspofungin- treated patients with candidemia liver enzyme elevations (ALT, AST or ALP) were the most frequent laboratory abnormalities (13.9%) and the most common clinical AE were fever (7.5%), chills (5.3%), nausea or vomiting (5.3%) and phlebitis (3.5%) [39]. Other reported infu- sion reactions to echinocandins in humans, including rash, pruritus, facial swelling and ana- phylaxis are mediated by histamine release [40]. Manifestations of acute anaphylaxis are species-dependent and directly related to the loca- tions of the largest population of mast cells, which are the heart and lungs in humans, gastroin- testinal tract and liver in dogs, and lungs in cats [41–48]. Thus, animal-models of disease need to account for such species differences and the effect of caspofungin in cats with IFIs needs to be evaluated in prospective studies. Echinocandins have concentration-dependent activity against Aspergillus spp [49]. In this study, at a dose of 1 mg/kg q 24 h Cmax and Cmin were well in excess of the highest MECs reported for Aspergillus viridinutans complex species. Caspofungin had MECs of less than 0.06 μg/mL for clinical isolates of A. felis from cats with invasive aspergillosis, except for one isolate in which the MEC was 2 μg/mL [1]. However, other cryptic species in section Fumigati reported to cause invasive aspergillosis in humans and cats have shown decreased susceptibil- ity to caspofungin, including some pathogenic isolates of A. lentulus which had MECs of cas- pofungin of 4 –>32 μg/mL [32]. In a murine model of invasive pulmonary aspergillosis caspofungin dose escalation and

dose fractionation was evaluated and Cmax: MEC ratio was the parameter most strongly associ- ated with reduced fungal burden, with an optimal value of 10–20. Mice dosed with 1 mg/kg q24 h or 2 mg/kg q48 h had a significantly lower fungal burdens than mice dosed with 1 mg/kg

q6 h [29]. At higher doses of 4 mg/kg, correlating to a Cmax: MEC >20 there was a paradoxical effect on fungal growth resulting in increased burdens in mice. This paradoxical effect on fun- gal growth of A. fumigatus has also been observed in vitro for caspofungin [49], and both in vitro and in vivo for other first and second generation echinocandins [50, 51]. Although not proven in clinical trials, the paradoxical growth effect of caspofungin at Cmax: MEC > 20 in vivo has led to concerns about possible reduced clinical efficacy using dosing

regimens where the Cmax greatly exceeds the MEC [29]. In this study, using a dose regimen of 1 mg/kg q24 h, Cmax: MEC was > 20 in 42.6% of cats after multiple doses. Our simulated data shows that an optimal Cmax: MEC of 10–20 is achieved using 0.75 mg/kg q 24 h or 1 mg/kg q 3 days. However, at the higher dose with a 3 day inter-dosing interval, Cmin is well below 1 μg/ mL in more than 95% of the population as the prolonged dosing interval does not allow for

drug accumulation. Using a daily dose of 0.75 mg/kg, the Cmax: MEC is below 20 and Cmin is greater than 2.5 μg/mL for more than 95% of the population. This difference in exposure between doses is also highlighted by the 50% reduction in the cumulative AUC above the MEC for the 1 mg/kg q 72 h regimen compared to 0.75 mg/kg q 24 h. Although both 1 mg/kg q 72 h

and 0.75 mg/kg q 24 h provide very similar results for Cmax: MEC, the latter regimen is vastly superior in regard to AUC > MEC. Thus, based on the modeling data, a daily dose of 0.75 mg/

kg daily is likely to best meet the proposed Cmax: MEC window, as well as provide more consis- tent exposure between doses. Alternatively, utilising a loading dose regimen of 1mg/kg on day 1, followed by 0.75mg/kg daily will rapidly achieve steady state, as well as provide continual exposure between dosing. The relationship between antifungal in vivo susceptibility, dosages, drug concentrations within various body compartments and toxicological effects are not completely understood. Although our pharmacokinetic analyses have attempted to predict a regimen appropriate for

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safe and effective use of caspofungin using in vitro MEC values, inherent biological factors can affect therapeutic outcome. For example, in sino-orbital aspergillosis systemic therapy may not be sufficient to penetrate the site of infection and reduce fungal burden. In addition, our work here is based upon total plasma concentrations, and not unbound drug, which may further confound the results. The echinocandins are highly protein bound, and this does not appear to vary across mammalian species investigated [14, 15, 33, 52]. The unique properties of caspo- fungin create a challenge in determining the precise level of protein binding [14]. However, studies have shown that caspofungin is bound to albumin in serum and that binding in mouse and human serum is equivalent, with an unbound fraction of caspofungin of 3–4%, which is concentration independent [14, 33]. Caspofungin has been shown to bind to albumin in serum [14, 33]. The plasma unbound fraction of caspofungin in the cat was not determined in this study, and although likely to be similar to that of humans and mice, based on similar albu- min concentrations in these three species, this should be confirmed in future investigations. The current regimen employed clinically provides total plasma concentrations consistent with

the Cmax:MEC ratio proposed in the present analyses, suggesting that our simulations are likely to be clinically relevant. Further pharmacodynamic studies are therefore required which can be linked with the pharmacokinetic data presented here, prior to a robust prospective clinical study to document clinical efficacy and safety of the proposed regimens in feline patients affected by sino-orbital aspergillosis.

Supporting information S1 Table. Summary of caspofungin simulations. (RTF)

Acknowledgments This study was funded by the Australian Companion Animal Health Foundation 009/2015. The caspofungin assay internal standard was supplied by Merck Sharp & Dohme Corp, 1 Merck Drive, Whitehouse Station, New Jersey 08889, USA. VB, AM and JL designed the study. JL, VB and RN obtained the experimental data. DJRF performed modeling and simula- tion of the data. All authors contributed to the writing of the manuscript. The Australian Cen- tre for Pharmacometrics is an initiative of the Australian Government as part of the National Collaborative Research Infrastructure Strategy.

Author Contributions Conceptualization: VB JL AM. Data curation: VB JL RN DF. Formal analysis: JL RN DF AM. Funding acquisition: VB. Investigation: JL VB RN. Methodology: RN DF. Project administration: VB. Resources: JL VB RN DF. Software: DF.

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Supervision: VB AM DF RN. Validation: DF AM FN. Visualization: DF AM. Writing – original draft: JL VB. Writing – review & editing: JL VB AM DF RN.

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41. Shmuel DL, Cortes Y. Anaphylaxis in dogs and cats. Journal of Veterinary Emergency and Critical Care. 2013; 23(4):377±94. https://doi.org/10.1111/vec.12066 PMID: 23855441 42. Litster A, Atwell R. Physiological and haematological findings and clinical observations in a model of acute systemic anaphylaxis in Dirofilaria immitis-sensitised cats. Australian veterinary journal. 2006; 84 (5):151±7. PMID: 16739523. 43. Richardson P, Withrington P. Responses of the simultaneously-perfused hepatic arterial and portal venous vascular beds of the dog to histamine and 5-hydroxytryptamine. British journal of pharmacology. 1978; 64(4):581±8. PMID: 728685 44. Dean H, Webb R. The morbid anatomy and histology of anaphylaxis in the dog. The Journal of Pathol- ogy and Bacteriology. 1924; 27(1):51±64. 45. Quantz JE, Miles MS, Reed AL, White GA. Elevation of alanine transaminase and gallbladder wall abnormalities as biomarkers of anaphylaxis in canine hypersensitivity patients. Journal of Veterinary Emergency and Critical Care. 2009; 19(6):536±44. https://doi.org/10.1111/j.1476-4431.2009.00474.x PMID: 20017759 46. Lautt WW, Legare DJ. Effect of histamine, norepinephrine, and nerves on vascular pressures in dog liver. American Journal of Physiology-Gastrointestinal and Liver Physiology. 1987; 252(4):G472±G8. PMID: 3565566. 47. Greenberger PA, Rotskoff BD, Lifschultz B. Fatal anaphylaxis: postmortem findings and associated comorbid diseases. Annals of allergy, asthma & immunology. 2007; 98(3):252±7. https://doi.org/10. 1016/S1081-1206(10)60714-4 48. Genovese A, Rossi FW, Spadaro G, Galdiero M, Marone G. Human cardiac mast cells in anaphylaxis. Anaphylaxis. 95: Karger Publishers; 2010. p. 98±109. 49. Lewis RE, Albert ND, Kontoyiannis DP. Comparison of the dose-dependent activity and paradoxical effect of caspofungin and micafungin in a neutropenic murine model of invasive pulmonary aspergillo- sis. Journal of antimicrobial chemotherapy. 2008; 61(5):1140±4. https://doi.org/10.1093/jac/dkn069 PMID: 18305201 50. Petraitiene R, Petraitis V, Groll AH, Sein T, Schaufele RL, Francesconi A, et al. Antifungal efficacy of caspofungin (MK-0991) in experimental pulmonary aspergillosis in persistently neutropenic rabbits: pharmacokinetics, drug disposition, and relationship to galactomannan antigenemia. Antimicrobial agents and chemotherapy. 2002; 46(1):12±23. PMID: 11751105 https://doi.org/10.1128/AAC.46.1.12- 23.2002 51. Petraitis V, Petraitiene R, Groll AH, Bell A, Callender DP, Sein T, et al. Antifungal efficacy, safety, and single-dose pharmacokinetics of LY303366, a novel echinocandin B, in experimental pulmonary asper- gillosis in persistently neutropenic rabbits. Antimicrobial agents and chemotherapy. 1998; 42(11):2898± 905. PMID: 9797223; 52. Sandhu P, Xu X, Bondiskey PJ, Balani SK, Morris ML, Tang YS, et al. Disposition of caspofungin, a novel antifungal agent, in mice, rats, rabbits, and monkeys. Antimicrobial agents and chemotherapy. 2004; 48(4):1272±80. PMID: 15047529 https://doi.org/10.1128/AAC.48.4.1272-1280.2004

PLOS ONE | https://doi.org/10.1371/journal.pone.0178783 June 2, 2017 14 / 14 Summary of Caspofungin simulations Each regimen has data provided for each dose of the 7 day regimen. This will be the 7th dose for the q 24h regimen and the 25th dose for the q 6h regimen. However, for the q6h and q12h regimens only data for the last dose each day is provided. Note that for dose regimens, there are several infusion durations (1h, 2h and 4h).

Dose regimen Period Cmax/MIC Dose Infusion Interval Duration Ratio > 10 10 > Ratio > 20 Dose (h) (h) Time (h) Dose Number (%) Ratio > 20 (%) (%) 0.25mg/kg q6 1h 1 First Dose 0 0 0 0.25mg/kg q6 1h 25 Fifth Dose 27.5 0 27.5 0.25mg/kg q6 1h 49 Ninth Dose 65.6 0 65.6 0.25mg/kg q6 1h 73 Thirteenth Dose 76.4 0 76.4 0.25mg/kg q6 1h 97 Seventeenth Dose 77.9 0 77.9 0.25mg/kg q6 1h 121 Twenty first Dose 78.5 0 78.5 0.25mg/kg q6 1h 145 Twenty fifth Dose 78.7 0 78.7 0.5mg/kg q12 1h 1 First Dose 2.40 0 2.40 0.5mg/kg q12 1h 25 Third Dose 88.6 0 88.6 0.5mg/kg q12 1h 49 Fifth Dose 95.5 0.600 94.9 0.5mg/kg q12 1h 73 Seventh Dose 97.5 2.00 95.5 0.5mg/kg q12 1h 97 Ninth Dose 97.7 2.60 95.1 0.5mg/kg q12 1h 121 Eleventh Dose 97.7 2.70 95.0 0.5mg/kg q12 1h 145 Thirteenth Dose 97.7 2.70 95.0 0.5mg/kg q24 1h 1 First Dose 2.10 0 2.10 0.5mg/kg q24 1h 25 Second Dose 25.2 0 25.2 0.5mg/kg q24 1h 49 Third Dose 44.3 0 44.3 0.5mg/kg q24 1h 73 Fourth Dose 44.3 0 44.3 0.5mg/kg q24 1h 97 Fifth Dose 44.3 0 44.3 0.5mg/kg q24 1h 121 Sixth Dose 44.3 0 44.3 0.5mg/kg q24 1h 145 Seventh Dose 44.3 0 44.3 0.5mg/kg q6 1h 1 First Dose 2.20 0 2.20 0.5mg/kg q6 1h 25 Fifth Dose 100 27.8 72.2 0.5mg/kg q6 1h 49 Ninth Dose 100 68.6 31.4 0.5mg/kg q6 1h 73 Thirteenth Dose 100 76.7 23.3 0.5mg/kg q6 1h 97 Seventeenth Dose 100 78.8 21.2 0.5mg/kg q6 1h 121 Twenty first Dose 100 79.5 20.5 0.5mg/kg q6 1h 145 Twenty fifth Dose 100 79.8 20.2 0.75mg/kg q24 1h 1 First Dose 70.9 0 70.9 0.75mg/kg q24 1h 25 Second Dose 97.4 0.300 97.1 0.75mg/kg q24 1h 49 Third Dose 98.4 1.60 96.8 0.75mg/kg q24 1h 73 Fourth Dose 98.4 1.60 96.8 0.75mg/kg q24 1h 97 Fifth Dose 98.5 1.70 96.8 0.75mg/kg q24 1h 121 Sixth Dose 98.5 1.80 96.7 0.75mg/kg q24 1h 145 Seventh Dose 98.5 1.90 96.6 0.75mg/kg q48 1h 1 First Dose 72.8 0 72.8 0.75mg/kg q48 1h 49 Second Dose 86.0 0 86.0 0.75mg/kg q48 1h 97 Third Dose 87.1 0 87.1 0.75mg/kg q48 1h 145 Third Dose 88.3 0 88.3 0.75mg/kg q72 1h 1 First Dose 72.6 0 72.6 0.75mg/kg q72 1h 73 Second Dose 79.0 0 79.0 0.75mg/kg q72 1h 145 Third Dose 80.9 0 80.9 1mg/kg q24 1h 1 First Dose 99.3 0.700 98.6 1mg/kg q24 1h 25 Second Dose 100 24.3 75.7 1mg/kg q24 1h 49 Third Dose 100 32.5 67.5 1mg/kg q24 1h 73 Fourth Dose 100 39.7 60.3 1mg/kg q24 1h 97 Fifth Dose 100 41.5 58.5 1mg/kg q24 1h 121 Sixth Dose 100 42.1 57.9 1mg/kg q24 1h 145 Seventh Dose 100 42.6 57.4 1mg/kg q24 2h 1 First Dose 1.60 0 1.60 1mg/kg q24 2h 25 Second Dose 96.1 0 96.1 1mg/kg q24 2h 49 Third Dose 99.8 6.50 93.3 1mg/kg q24 2h 73 Fourth Dose 100 14.6 85.4 1mg/kg q24 2h 97 Fifth Dose 100 17.7 82.3 1mg/kg q24 2h 121 Sixth Dose 100 19.3 80.7 1mg/kg q24 2h 145 Seventh Dose 100 20.0 80.0 1mg/kg q24 4h 1 First Dose 0 0 0 1mg/kg q24 4h 25 Second Dose 80.4 0 80.4 1mg/kg q24 4h 49 Third Dose 99.3 0.700 98.6 1mg/kg q24 4h 73 Fourth Dose 99.5 2.70 96.8 1mg/kg q24 4h 97 Fifth Dose 99.5 3.80 95.7 1mg/kg q24 4h 121 Sixth Dose 99.5 4.60 94.9 1mg/kg q24 4h 145 Seventh Dose 99.5 5.40 94.1 1mg/kg q48 1h 1 First Dose 99.8 2.10 97.7 1mg/kg q48 1h 49 Second Dose 99.9 5.70 94.2 1mg/kg q48 1h 97 Third Dose 99.9 5.70 94.2 1mg/kg q48 1h 145 Third Dose 99.9 5.80 94.1 1mg/kg q48 2h 1 First Dose 0.900 0 0.900 1mg/kg q48 2h 49 Second Dose 96.1 0 96.1 1mg/kg q48 2h 97 Third Dose 97.8 0.200 97.6 1mg/kg q48 2h 145 Third Dose 98.2 0.300 97.9 1mg/kg q48 4h 1 First Dose 0 0 0 1mg/kg q48 4h 49 Second Dose 79.2 0 79.2 1mg/kg q48 4h 97 Third Dose 90.1 0 90.1 1mg/kg q48 4h 145 Third Dose 91.4 0 91.4 1mg/kg q72 1h 1 First Dose 99.1 1.70 97.4 1mg/kg q72 1h 73 Second Dose 99.2 2.80 96.4 1mg/kg q72 1h 145 Third Dose 99.2 3.80 95.4 1mg/kg q72 2h 1 First Dose 2.20 0 2.20 1mg/kg q72 2h 73 Second Dose 96.4 0 96.4 1mg/kg q72 2h 145 Third Dose 97.2 0.300 96.9 1mg/kg q72 4h 1 First Dose 0 0 0 1mg/kg q72 4h 73 Second Dose 80.8 0 80.8 1mg/kg q72 4h 145 Third Dose 84.0 0 84.0 2mg/kg q48 1h 1 First Dose 100 99.6 0.400 2mg/kg q48 1h 49 Second Dose 100 99.7 0.300 2mg/kg q48 1h 97 Third Dose 100 99.7 0.300 2mg/kg q48 1h 145 Third Dose 100 99.7 0.300 2mg/kg q48 2h 1 First Dose 100 1.50 98.5 2mg/kg q48 2h 49 Second Dose 100 96.6 3.40 2mg/kg q48 2h 97 Third Dose 100 99.4 0.600 2mg/kg q48 2h 145 Third Dose 100 99.4 0.600 2mg/kg q48 4h 1 First Dose 2.20 0 2.20 2mg/kg q48 4h 49 Second Dose 100 76.7 23.3 2mg/kg q48 4h 97 Third Dose 100 90.1 9.90 2mg/kg q48 4h 145 Third Dose 100 91.0 9.00 2mg/kg q72 1h 1 First Dose 100 98.9 1.10 2mg/kg q72 1h 73 Second Dose 100 99.4 0.600 2mg/kg q72 1h 145 Third Dose 100 99.4 0.600 2mg/kg q72 2h 1 First Dose 99.0 1.00 98.0 2mg/kg q72 2h 73 Second Dose 100 94.7 5.30 2mg/kg q72 2h 145 Third Dose 100 95.9 4.10 2mg/kg q72 4h 1 First Dose 1.80 0 1.80 2mg/kg q72 4h 73 Second Dose 100 78.1 21.9 2mg/kg q72 4h 145 Third Dose 100 82.2 17.8 102

Appendix 2

S-WPC-MK0991-IV-102016 1

PRODUCT INFORMATION

CANCIDAS ® (caspofungin acetate)

DESCRIPTION

CANCIDAS® is a sterile, lyophilised product for intravenous infusion that contains a semisynthetic lipopeptide (echinocandin) compound synthesised from a fermentation product of Glarea lozoyensis. CANCIDAS is the first of a new class of antifungal drugs (echinocandins) that inhibit the synthesis of  (1,3)-D-glucan, an integral component of the fungal cell wall.

CANCIDAS (caspofungin acetate) is 1-[(4R,5S)-5-[(2-aminoethyl)amino]-N2-(10,12- dimethyl-1-oxotetradecyl)-4-hydroxy-L-ornithine]-5-[(3R)-3-hydroxy-L- ornithine]pneumocandin B0 diacetate (salt). In addition to the active ingredient caspofungin acetate, CANCIDAS contains the following inactive ingredients: sucrose, mannitol, acetic acid, glacial, and sodium hydroxide. Caspofungin acetate is a hygroscopic, white to off-white powder. It is freely soluble in water and methanol, and slightly soluble in ethanol. The pH of a saturated aqueous solution of caspofungin acetate is approximately 6.6. The empirical formula is C52H88N10O15•2C2H4O2 and the formula weight is 1213.42. The CAS No is 179463- 17-3. The structural formula is:

H2N NH OH O O HO N H H N

H2N N O H3C HN OH O CH3 CH3

HO NH O CH3 O H N •2CH CO H N 3 2 HO OH O OH

HO

PHARMACOLOGY

Mechanism of Action Caspofungin acetate, the active ingredient of CANCIDAS, inhibits the synthesis of  (1,3)-D-glucan, an essential component of the cell wall of many filamentous fungi and yeast.  (1,3)-D-glucan is not present in mammalian cells.

Microbiology Activity in vitro Caspofungin has in vitro activity against: Aspergillus species (including Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus and Aspergillus candidus) Candida species (including Candida albicans, Candida dublinensis, Candida glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lipolytica, Candida lusitaniae, Candida parapsilosis, Candida rugosa, and Candida tropicalis).

S-WPC-MK0991-IV-102016 2

Susceptibility testing was performed according to a modification of both the Clinical and Laboratory Standards Institute (CLSI, formerly known as the National Committee for Clinical Laboratory Standards (NCCLS) method M38-A2 (for Aspergillus species) and method M27-A3 (for Candida species).

Interpretive standards (or breakpoints) for caspofungin against Candida species are applicable only to tests performed using CLSI microbroth dilution reference method M27-A3 for minimum inhibitory concentrations (MIC) read as a partial inhibition endpoint at 24 hours. The MIC values for caspofungin using CLSI microbroth dilution reference method M27-A3 should be interpreted according to the criteria provided in Table 1 below (CLSI M27-S3).

TABLE 1 Susceptibility Interpretive Criteria for Caspofungin against Candida species

Broth Microdilution MIC*, † (g/mL) at 24 Pathogen hours Susceptible Indeterminate Resistant Non-susceptible Candida species 2 - - ≥2 * A report of “Susceptible” indicates that the pathogen is likely to be inhibited if the antimicrobial compound in the blood reaches the concentrations usually achievable. † There is no Resistant category assigned for the echinocandin agents; isolates with higher MICs may be described as Non-susceptible.

There are no established breakpoints for caspofungin against Candida species using the European Committee for Antimicrobial Susceptibility Testing (EUCAST) method.

Standardised techniques for susceptibility testing have been established for yeasts by EUCAST. No standardised techniques for susceptibility testing or interpretive breakpoints have been established for Aspergillus species and other filamentous fungi using either the CLSI or EUCAST method.

Activity in vivo Caspofungin was active when parenterally administered to immune-competent and immune-suppressed animals with disseminated infections of Aspergillus and Candida for which the endpoints were prolonged survival of infected animals (Aspergillus and Candida) and clearance of fungi from target organs (Candida). Caspofungin was also active in immunodeficient animals after disseminated infection with C. glabrata, C. krusei, C. lusitaniae, C. parapsilosis, or C. tropicalis in which the endpoint was clearance of Candida from target organs. Caspofungin has been reported to be active in the prevention and treatment of pulmonary aspergillosis in a rat model.

Cross-resistance Caspofungin acetate is active against strains of Candida with intrinsic or acquired resistance to fluconazole, amphotericin B, or flucytosine consistent with their different mechanisms of action.

Drug Resistance A caspofungin MIC of 2 g/mL (“Susceptible” per Table 1) using the CLSI M27-A3 method indicates that the Candida isolate is likely to be inhibited if caspofungin therapeutic concentrations are achieved. Breakthrough infections with Candida isolates requiring caspofungin concentrations 2 g/mL for growth inhibition have developed in a mouse model of C. albicans infection. Isolates of Candida with reduced susceptibility to caspofungin have been identified in a small number of patients during treatment (MICs for caspofungin >2 g/mL using standardised MIC S-WPC-MK0991-IV-102016 3 testing techniques approved by the CLSI). Some of these isolates had mutations in the FKS1/FKS2 gene. Although the incidence is rare, these cases have been routinely associated with poor clinical outcomes.

Development of in vitro resistance to caspofungin by Aspergillus species has been identified. In clinical experience, drug resistance in patients with invasive aspergillosis has been observed. The mechanism of resistance has not been established.

The incidence of drug resistance in various clinical isolates of Candida and Aspergillus species is rare.

Pharmacokinetics

Absorption Absorption is not relevant since caspofungin acetate is administered intravenously.

Distribution Plasma concentrations of caspofungin decline in a polyphasic manner following single 1-hour IV infusions. A short -phase occurs immediately post-infusion, followed by a -phase (half-life of 9 to 11 hours) that characterises much of the profile and exhibits clear log-linear behaviour from 6 to 48 hours post-dose (during which the plasma concentration decreases by an order of magnitude). An additional, longer half-life (-) phase, also occurs with a half-life of 40-50 hours.

Distribution, rather than excretion or biotransformation, is the dominant mechanism influencing plasma clearance. Caspofungin is extensively bound to albumin (~97%), and distribution into red blood cells is minimal. Mass balance results showed that approximately 92% of the [3H] label was found in tissues 36 to 48 hours after a single 70 mg dose of [3H] caspofungin acetate. There is little excretion or biotransformation of caspofungin during the first 30 hours after administration.

Metabolism Caspofungin is slowly metabolised by hydrolysis and N-acetylation. Caspofungin also undergoes spontaneous chemical degradation to an open-ring peptide compound, L-747969. At later time points (5 days post-dose), there is a low level ( 7 picomoles/mg protein, or  1.3% of administered dose) of covalent binding of radiolabel in plasma, following single-dose administration of [3H] caspofungin acetate. This may be due to two reactive intermediates formed during the chemical degradation of caspofungin to L-747969. Additional metabolism involves hydrolysis into constitutive amino acids and their degradates, including dihydroxyhomotyrosine and N-acetyl-dihydroxyhomotyrosine. These two tyrosine derivatives are found only in urine, suggesting rapid clearance of these derivatives by the kidneys.

Excretion Two single-dose radiolabelled pharmacokinetic studies were conducted. In one study, plasma, urine, and faeces were collected over 27 days, and in the second study, plasma was collected over six months. Approximately 75% of the radioactivity was recovered: 41% in urine and 34% in faeces. Plasma concentrations of radioactivity and of caspofungin were similar during the first 24 to 48 hours post- dose; thereafter drug levels fell more rapidly. In plasma, caspofungin concentrations fell below the limit of quantitation after 6 to 8 days post-dose, while radio-label fell S-WPC-MK0991-IV-102016 4 below the limit of quantitation at 22.3 weeks post-dose. A small amount of caspofungin is excreted unchanged in urine (~1.4% of dose). Renal clearance of parent drug is low (~0.15 mL/min).

Special Populations Gender The plasma concentration of caspofungin was similar in healthy men and women on Day 1 following a single 70 mg dose. After 13 daily 50 mg doses, the area under the curve (AUC) for caspofungin was elevated slightly (approximately 20%) in women relative to men. No dosage adjustment is necessary based on gender.

Geriatric Plasma concentrations of caspofungin in healthy older men and women (65 years of age) were increased slightly (approximately 28% in area under the curve [AUC]) compared to young healthy men. In patients who were treated empirically or who had invasive candidiasis, a similar modest effect of age was seen in older patients relative to younger patients. No dosage adjustment is necessary for the elderly.

Race No clinically significant differences in the pharmacokinetics of caspofungin were seen among Caucasians, Blacks, Hispanics, and persons of mixed race. No dosage adjustment is necessary on the basis of race.

Renal Insufficiency In a clinical study of single 70 mg doses, caspofungin pharmacokinetics were similar in volunteers with mild renal insufficiency (creatinine clearance 50 to 80 mL/min) and control subjects. Moderate (creatinine clearance 31 to 49 mL/min), advanced (creatinine clearance 5 to 30 mL/min), and end-stage (creatinine clearance <10 mL/min and dialysis dependent) renal insufficiency moderately increased caspofungin plasma concentrations after single-dose administration (range: 30 to 49% for AUC). However, in patients with invasive aspergillosis who received multiple daily doses of CANCIDAS 50 mg, there was no significant effect of mild to advanced renal impairment on caspofungin trough concentrations. No dosage adjustment is necessary for patients with renal insufficiency. Caspofungin is not dialysable, thus supplementary dosing is not required following haemodialysis.

Hepatic Insufficiency Plasma concentrations of caspofungin after a single 70 mg dose in patients with mild hepatic insufficiency (Child-Pugh score 5 to 6) were increased by approximately 55% in AUC compared to healthy control subjects. In a 14-day multiple-dose study (70 mg on Day 1 followed by 50 mg daily thereafter), plasma concentrations in patients with mild hepatic insufficiency were increased modestly (21 to 26% in AUC) on Days 7 and 14 relative to healthy control subjects. No dosage adjustment is recommended for patients with mild hepatic insufficiency. Patients with moderate hepatic insufficiency (Child-Pugh score 7 to 9) who received a single 70 mg dose of CANCIDAS had an average plasma caspofungin increase of 76% compared to control subjects.

A dosage reduction is recommended for patients with moderate hepatic insufficiency (see DOSAGE AND ADMINISTRATION). There is no clinical experience in patients with severe hepatic insufficiency (Child-Pugh score >9).

S-WPC-MK0991-IV-102016 5

Paediatric Patients CANCIDAS has been studied in five prospective studies involving paediatric patients under 18 years of age, including three paediatric pharmacokinetic studies (initial study in adolescents [12-17 years of age] and children [2-11 years of age] followed by a study in younger patients [3-23 months of age] and then followed by a study in neonates and infants [<3 months]).

 In adolescents (ages 12 to 17 years) receiving caspofungin at 50 mg/m2 daily (maximum 70 mg daily), the caspofungin plasma AUC0-24hr was generally comparable to that seen in adults receiving caspofungin at 50 mg daily. All adolescents received doses >50 mg daily, and, in fact, 6 of 8 received the maximum dose of 70 mg/day. The caspofungin plasma concentrations in these adolescents were reduced relative to adults receiving 70 mg daily, the dose most often administered to adolescents.  In children (ages 2 to 11 years) receiving caspofungin at 50 mg/m2 daily (maximum 70 mg daily), the caspofungin plasma AUC0-24hr after multiple doses was comparable to that seen in adults receiving caspofungin at 50 mg/day. On the first day of administration, AUC0-24hr was somewhat higher in children than adults for these comparisons (37% increase for the 50 mg/m2/day to 50 mg/day comparison).  In young children and toddlers (ages 3 to 23 months) receiving caspofungin at 50 2 mg/m daily (maximum 70 mg daily), the caspofungin plasma AUC0-24hr after multiple doses was comparable to that seen in adults receiving caspofungin at 50 mg daily. As in the older children, these young children who received 50 mg/m2 daily had slightly higher AUC0-24 hr values on Day 1 relative to adults receiving the standard 50-mg daily dose. The caspofungin pharmacokinetic results from the young children (3 to 23 months of age) that received 50 mg/m2 caspofungin daily were similar to the pharmacokinetic results from older children (2 to 11 years of age) that received the same dosing regimen.  In neonates and infants (<3 months) receiving caspofungin at 25 mg/m2 daily, caspofungin peak concentration (C1 hr) and caspofungin trough concentration (C24 hr) after multiple doses were comparable to that seen in adults receiving caspofungin at 50 mg daily. On Day 1, C1 hr was comparable and C24 hr modestly elevated (36%) in these neonates and infants relative to adults. AUC0-24hr measurements were not performed in this study due to the sparse plasma sampling. Of note, the efficacy and safety of CANCIDAS has not been adequately studied in prospective clinical trials involving neonates and infants under 3 months of age.

Based on population pharmacokinetic analyses including data from the 2 efficacy studies in paediatric population and in comparison with healthy adults and adult patients, the caspofungin C1 hr was significantly increased (50 – 163%) in paediatric patients of all age groups, whereas C24 hr was significantly increased (25-109%) in older children (2 – 11 years) and adolescents (12 – 17 years), but was similar to adults in young children (3 – 24 months). The estimated increase in AUC0-24 hr was 38 – 41% in paediatric patients of all age groups compared with adult patients.

Overall, the available pharmacokinetic, efficacy, and safety data are limited in patients 3 to 10 months of age. Pharmacokinetic data from one 10-month old child 2 receiving the 50 mg/m daily dose indicated an AUC0-24hr within the same range as that observed in older children and adults at the 50 mg/m2 and the 50 mg dose, 2 respectively, while in one 6-month old child receiving the 50 mg/m dose, the AUC0- 24hr was somewhat higher.

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Clinical Trials

The results of the adult clinical studies are presented by each indication below, followed thereafter by the results of paediatric clinical trials.

Empirical Therapy in febrile, neutropenic patients A multicentre, double-blind study enrolled 1111 febrile, neutropenic, adult patients (mean age 48 years, range 16-83; 56% male) who were randomised to treatment with daily doses of CANCIDAS (50 mg/day following a 70 mg loading dose on Day 1) or AmBisome®1 (liposomal amphotericin for injection, 3.0 mg/kg/day). Eligible patients had received chemotherapy for malignancy or had undergone haematopoietic stem-cell transplantation (HSCT), and presented with neutropenia (<500 cells/mm3 for 96 hours) and fever (>38.0°C) that had not responded to antibacterial therapy. Any patient known to have a documented fungal infection was excluded from entering the study. Patients were to be treated until resolution of neutropenia, with a maximum treatment duration of 28 days. However, patients found to have a documented fungal infection could be treated longer. If the drug was well tolerated but the patient’s fever persisted and clinical condition deteriorated following 5 days of therapy, the dosage of study drug could be increased to 70 mg/day for CANCIDAS (13.3% of patients treated) or to 5.0 mg/kg/day for AmBisome (14.3% of patients treated).

Patients were stratified based on risk category [high-risk patients had undergone allogeneic HSCT (7.0% total) or had relapsed acute leukaemia (17.7% total)] and on receipt of prior antifungal prophylaxis. The percentage of patients in the high-risk stratum at entry was 26.6% for the CANCIDAS group and 22.9% for the AmBisome group. In both groups a similar percentage of patients had received antifungal prophylaxis. The most frequent diagnoses were acute myelogenous leukaemia, acute lymphocytic leukaemia, and non-Hodgkin’s lymphoma.

Patients who met the entry criteria and received at least one dose of study therapy were included in the modified intention-to-treat (MITT) population (556 treated with CANCIDAS and 539 treated with AmBisome). The mean duration of study therapy was 13 days. An overall favourable response required meeting each of 5 criteria: 1) successful treatment of any baseline fungal infection, 2) no breakthrough fungal infections during administration of study drug or within 7 days after completion of treatment, 3) survival for 7 days after completion of study therapy, 4) no discontinuation of the study drug because of drug-related toxicity or lack of efficacy, and 5) resolution of fever during the period of neutropenia.

An independent expert panel adjudicated blinded data from all patients identified as having a suspected invasive fungal infection. The panel assessed the presence of invasive fungal infection, timing of onset (baseline or breakthrough), causative pathogen, and, for baseline infections, response to study treatment. The only fungal infections considered to be present for purposes of statistical analysis were those classified by the expert panel as either probable or proven. Approximately 5% of patients were found to have baseline fungal infections, of which the majority were due to Aspergillus or Candida species.

The proportion of MITT patients with an overall favourable response and the proportion of MITT patients with favourable responses to the individual criteria are shown in Table 2.

1 Registered trademark of Gilead Sciences, Inc. S-WPC-MK0991-IV-102016 7

Table 2 Favourable Response of Patients with Persistent Fever and Neutropenia % Difference CANCIDAS* AmBisome* (Confidence Interval) ** Number of Patients (Modified Intention- 556 539 To-Treat) Overall Favourable Response 190 (33.9%) 181 (33.7%) 0.2 (-5.6, 6.0) 1. Successful treatment of any baseline 14/27 7/27 25.9 (0.9, 51.0)† fungal infection (51.9%) (25.9%) 2. No breakthrough fungal infection 527 (94.8%) 515 (95.5%) -0.8 (-3.3, 1.8) 3. Survival 7 days after end of treatment 515 (92.6%) 481 (89.2%) 3.4 (0.0, 6.8) 4. No discontinuation due to toxicity or 499 (89.7%) 461 (85.5%) 4.2 (0.3, 8.1)† lack of efficacy 5. Resolution of fever during neutropenia 229 (41.2%) 223 (41.4%) -0.2 (-6.0, 5.6) * CANCIDAS: 70 mg on Day 1, then 50 mg daily for the remainder of treatment (daily dose increased to 70 mg for 73 patients); AmBisome: 3.0 mg/kg/day (daily dose increased to 5.0 mg/kg for 74 patients). ** Overall Response (primary efficacy endpoint): estimated % difference adjusted for strata and expressed as CANCIDAS – AmBisome (95.2% CI);, based on all 5 criteria Individual Criteria: % difference calculated as CANCIDAS – AmBisome (95% CI). † Statistically significant difference.

Based on overall favourable response rates, CANCIDAS was as effective as AmBisome in empirical therapy of persistent febrile neutropenia. CANCIDAS had significantly higher favourable response rates than AmBisome for the following criteria: successful treatment of any baseline fungal infection (CANCIDAS 51.9%, AmBisome 25.9%) and absence of premature discontinuation from study therapy due to toxicity or lack of efficacy (CANCIDAS 89.7%, AmBisome 85.5%). CANCIDAS was comparable to AmBisome for the other criteria (absence of a breakthrough fungal infection, survival for 7 days after the end of treatment, and resolution of fever during neutropenia).

Overall favourable response rates were comparable in high-risk patients (CANCIDAS 43.2%, AmBisome 37.7%) and low-risk patients (CANCIDAS 31.0%, AmBisome 32.4%). Rates were also comparable in patients who had received prior antifungal prophylaxis (CANCIDAS 33.5%, AmBisome 32.9%) and those who had not (CANCIDAS 35.0%, AmBisome 34.5%).

The majority of baseline infections were due to Aspergillus or Candida species. Response rates to CANCIDAS and AmBisome for baseline infections caused by Aspergillus species were, respectively, 41.7% (5/12) and 8.3% (1/12), and by Candida species were 66.7% (8/12) and 41.7% (5/12).

Invasive Candidiasis In an initial Phase III randomised, double-blind study, patients with a proven diagnosis of invasive candidiasis received daily doses of CANCIDAS (50 mg/day following a 70-mg loading dose on Day 1) or amphotericin B deoxycholate (0.6 to 0.7 mg/kg/day for non-neutropenic patients and 0.7 to 1.0 mg/kg/day for neutropenic patients). Patients were stratified by both neutropenic status and APACHE II score. Patients who met the entry criteria and received one or more doses of IV study therapy were included in the primary (modified intention-to-treat [MITT]) analysis of response at the end of IV study therapy. A predefined analysis to support the MITT, the evaluable-patients assessment, included patients who met entry criteria, received IV study therapy for 5 or more days and had a full efficacy evaluation at the end of IV S-WPC-MK0991-IV-102016 8

study therapy. A favourable response required both symptom resolution and microbiological clearance of the Candida infection.

Of the 239 patients enrolled, 224 (109 treated with CANCIDAS and 115 treated with amphotericin B) met the criteria for inclusion in the MITT analysis. Of these patients, 185 (88 treated with CANCIDAS and 97 treated with amphotericin B) met the criteria for inclusion in the evaluable-patients analysis. The most frequent diagnoses were bloodstream infections (candidaemia) (83%) and Candida peritonitis (10%), patients with Candida endocarditis, osteomyelitis or meningitis were excluded from this study. Most infections were caused by C. albicans (45%), followed by C. parapsilosis (19%), C. tropicalis (16%), C. glabrata (11%), and C. krusei (2%). The favourable response rates at the end of IV study therapy are shown in Table 3.

Table 3 Favourable Response Rates to IV Study Therapy Among Patients with Invasive Candidiasis (and Candidaemia)

CANCIDAS 50 mg* Amphotericin B Difference (%) after % (n/m**) % (n/m) Adjusting for strata [95% CI] [95% CI] [95.6% CI] ALL PATIENTS WITH INVASIVE CANDIDIASIS

MITT analysis 73.4% (80/109) 61.7% (71/115) 12.7% [65.1, 81.7] [52.8, 70.7] [-0.7, 26.0]

Neutropenic patients 50% (7/14) 40% (4/10) (ANC  500L)

APACHE II scores 57.1% (12/21) 43.5% (10/23) >20 at study entry

Evaluable-patients analysis 80.7% (71/88) 64.9% (63/97) 15.4% [72.4, 89.0] [55.4, 74.5] [1.1, 29.7]

PATIENTS WITH CANDIDAEMIA

MITT analysis 71.7% (66/92) 62.8% (59/94) 10.0% [62.5, 81.0] [52.9, 72.6] [-4.5, 24.5]

Evaluable-patients analysis 80.3% (57/71) 64.6% (51/79) 15.2% [71.0, 89.6] [53.9, 75.2] [-0.6, 31.0]

* Patients received CANCIDAS 70 mg on Day 1, then 50 mg daily for the remainder of their treatment. ** Number of patients with favourable response at the end of IV study therapy/number of patients included in analysis

Response rates were also consistent across all identified Candida species. For all other efficacy time points (Day 10 of IV study therapy, end of all antifungal therapy, 2- week post-therapy follow-up, and 6- to 8-week post-therapy follow-up), CANCIDAS was as effective as amphotericin B. CANCIDAS was also comparable to amphotericin B with regard to relapse or survival rates, with an overall mortality among MITT patients during the study treatment period and 6 to 8-week follow-up period of 33.0% in the CANCIDAS group and 30.4% in the amphotericin B group. CANCIDAS was comparable to amphotericin B in the treatment of invasive candidiasis at the end of IV study therapy in the primary (MITT) efficacy analysis. In a predefined efficacy analysis of evaluable patients to support the MITT, CANCIDAS was statistically superior to amphotericin B at the end of IV study therapy.

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Of the 224 patients from the invasive candidiasis study who met the criteria for inclusion in the MITT analysis, 186 patients (92 treated with CANCIDAS and 94 treated with amphotericin B) had candidaemia. Of these patients, 150 (71 treated with CANCIDAS and 79 treated with amphotericin B) met the criteria for inclusion in the evaluable-patients analysis. The favourable response rates at the end of IV study therapy for patients with candidaemia are shown in Table 3. In both the MITT and evaluable-patients efficacy analyses, CANCIDAS was comparable to amphotericin B in the treatment of candidaemia at the end of IV study therapy.

In a second Phase III randomised, double-blind study, patients with a proven diagnosis of invasive candidiasis received daily doses of CANCIDAS 50 mg/day (following a 70-mg loading dose on Day 1) or CANCIDAS 150 mg/day. The diagnostic criteria, efficacy time points, and efficacy endpoints used in this study were similar to those employed in the prior study. Efficacy was a secondary endpoint in this study. Patients who met the entry criteria and received one or more doses of caspofungin study therapy were included in the efficacy analysis. The favorable overall response rates at the end of CANCIDAS therapy were similar in the 2 treatment groups: 72% (73/102) and 78% (74/95) for the CANCIDAS 50-mg and 150- mg treatment groups, respectively (difference 6.3% [95% CI -5.9, 18.4]).

Oesophageal Candidiasis A total of 393 patients with oesophageal candidiasis were enrolled in three comparative studies to evaluate the efficacy of CANCIDAS for the treatment of oesophageal candidiasis. Patients were required to have symptoms and microbiological documentation of oesophageal candidiasis; and most patients were significantly immunocompromised. Disease severity was determined by oesophagoscopy (endoscopy).

In the randomised, double-blind study comparing CANCIDAS 50 mg/day (n=83) versus IV fluconazole 200 mg/day (n=94) for the treatment of oesophageal candidiasis, patients were treated for 7 to 21 days. A favourable overall response required both complete resolution of symptoms and significant endoscopic improvement 5 to 7 days following discontinuation of study therapy. The definition of endoscopic response was based on severity of disease at baseline using a 4 grade scale and required at least a two grade reduction from baseline endoscopic score or reduction to grade 0 for patients with a baseline score of 2 or less. The proportion of patients with a favourable response to CANCIDAS was comparable to that seen with fluconazole, as demonstrated in Table 4.

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Table 4 Oesophageal Candidiasis study: CANCIDAS v fluconazole Proportion of patients with a favourable response

CANCIDAS 50 mg Fluconazole 200 mg Observed Difference n/m (%) n/m (%) (%) [95% Confidence Interval] [95% Confidence Interval] [95% Confidence Interval] Favourable Overall 66/81 (81.5) 80/94 (85.1) -3.6 Combined Response [71.3, 89.2] [76.3, 91.6] [-14.7, 7.5] (Symptoms and Endoscopy

Symptom Response 73/81 (90.1) 84/94 (89.4) 0.8 [81.5, 95.6] [81.3, 94.8] [-8.2, 9.8]

Endoscopy Response 69/81 (85.2) 81/94 (86.2) -1.0 [75.6, 92.1] [77.5, 92.4] [-11.4, 9.4]

*n/m=number of patients with a favourable assessment/patients with data at 5 to 7 day follow-up visit

In addition, two double-blind, comparative dose-ranging studies evaluated 3 different doses of CANCIDAS (35, 50, 70 mg/day) and amphotericin B (0.5 mg/kg/day). These clinical studies support the use of CANCIDAS 50 mg daily in the treatment of oesophageal candidiasis. Increasing doses of CANCIDAS above 50 mg daily provided no additional benefit in the treatment of oesophageal candidiasis.

Invasive Aspergillosis Sixty nine patients between the ages of 18 and 80 with invasive aspergillosis were enrolled in an open-label, non-comparative study to evaluate the safety, tolerability, and efficacy of CANCIDAS. Enrolled patients had previously been refractory to or intolerant of other antifungal therapy. Refractory patients were classified as those who had disease progression or failed to improve despite therapy for at least 7 days with amphotericin B, lipid formulations of amphotericin B, itraconazole, or an investigational azole (voriconazole) with reported activity against Aspergillus. Intolerance to previous therapy was defined as a doubling of creatinine (or creatinine > 2.5 mg/dL while on therapy), other acute reactions, or infusion-related toxicity.

To be included in the study, patients with pulmonary disease must have had definite (positive tissue histopathology or positive culture from tissue obtained by an invasive procedure) or probable (positive radiographic or computed tomographic evidence with supporting culture from bronchoalveolar lavage or sputum, galactomannan enzyme-linked immunosorbent assay, and/or polymerase chain reaction) invasive aspergillosis.

Patients with extrapulmonary disease had to have definite invasive aspergillosis. The definitions were modelled after the Mycoses Study Group Criteria. Patients were administered a single 70 mg loading dose of CANCIDAS and subsequently dosed with 50 mg daily. The mean duration of therapy was 33.7 days, with a range of 1 to 162 days.

An independent expert panel evaluated patient data, including diagnosis of invasive aspergillosis, response and tolerability to previous antifungal therapy, treatment course on CANCIDAS, and clinical outcome.

A favourable response was defined as either complete resolution (complete response) or clinically meaningful improvement (partial response) of all signs and S-WPC-MK0991-IV-102016 11 symptoms and attributable radiographic findings. Stable, nonprogressive disease was considered to be an unfavourable response.

Among the 69 patients enrolled in the study, 63 met entry diagnostic criteria and had outcome data; and of these, 52 patients received treatment for >7 days. Fifty-three (84%) were refractory to previous antifungal therapy and 10 (16%) were intolerant. Forty five patients had pulmonary disease and 18 had extrapulmonary disease. Underlying conditions were haematologic malignancy (N=24), allogeneic bone marrow transplant or stem cell transplant (N=18), organ transplant (N=8), solid tumour (N=3), or other conditions (N=10). Fourteen patients were neutropenic (ANC < 500/L) at baseline; two of these patients had a favourable response to caspofungin therapy and evidence of their clinical response was seen prior to recovery of their neutrophil counts. In addition, 3 patients who had become neutropenic during caspofungin therapy all had a favourable response. None of the 7 patients who were persistently neutropenic had a favourable response.

All patients in the study received concomitant therapies for their other underlying conditions. Eighteen patients received tacrolimus and CANCIDAS concomitantly, (of whom 8 also received mycophenolate mofetil); 5 of these 18 patients had a favourable response. Among the 23 patients receiving high-dose corticosteroids, 8 had a favourable response, including 5 who continued on high-dose steroids. Overall, the expert panel determined that 41% (26/63) of patients receiving at least one dose of CANCIDAS had a favourable response. For those patients who received >7 days of therapy with CANCIDAS, 50% (26/52) had a favourable response. The favourable response rates for patients who were either refractory to or intolerant of previous therapies were 36% (19/53) and 70% (7/10), respectively. The response rates among patients with pulmonary disease and extrapulmonary disease were 47% (21/45) and 28% (5/18), respectively. Among patients with extrapulmonary disease, 2 of 8 patients who also had definite, probable, or possible CNS involvement had a favourable response.

A medical chart review of 206 patients with invasive aspergillosis was also conducted to assess the response to standard (non-investigational) therapies. Patient characteristics and important risk factors in this review were similar to those of patients enrolled in the open-label non-comparative study (see above), and the same rigorous definitions of diagnosis and outcome were used. To be included in this study, patients had to have had invasive aspergillosis and to have received at least 7 days of standard antifungal therapy. The favourable response rate from this historical control study was 17% (35/206) for standard therapy compared to the favourable response rate of 41% (26/63) for CANCIDAS in the open-label non- comparative study (the odds ratio was approximately 3).

Paediatric Patients The safety and efficacy of CANCIDAS was evaluated in paediatric patients 3 months to 17 years of age in two prospective, multicentre clinical trials.

The first study, which enrolled 82 patients between 2 to 17 years of age, was a randomised, double-blind study comparing CANCIDAS (50 mg/m2 IV once daily following a 70-mg/m2 loading dose on Day 1 [not to exceed 70 mg daily]) to AmBisome (3 mg/kg IV daily) in a 2:1 treatment fashion (56 on caspofungin, 26 on AmBisome) as empirical therapy in paediatric patients with persistent fever and neutropenia. The study design and criteria for efficacy assessment were similar to the study in adult patients (see Clinical Trials, Empirical Therapy in febrile, neutropenic patients). Patients were stratified based on risk category (high-risk patients had undergone allogeneic stem cell transplantation or had relapsed acute S-WPC-MK0991-IV-102016 12 leukaemia). Twenty-seven percent of patients in both treatment groups were high risk. The overall success rates in the MITT analysis, adjusted for strata, were as follows: 46% (26/56) for CANCIDAS and 32% (8/25) for AmBisome. For those patients in the high risk category, the favourable overall response rate was 60% (9/15) in the CANCIDAS group and 0% (0/7) in the AmBisome group.

The second study was a prospective, open-label, non-comparative study estimating the safety and efficacy of caspofungin in paediatric patients (ages 3 months to 17 years) with invasive candidiasis, oesophageal candidiasis, and invasive aspergillosis (as salvage therapy). The study employed diagnostic criteria which were based on established EORTC/MSG criteria of proven or probable infection; these criteria were similar to those criteria employed in the adult studies for these various indications. Similarly, the efficacy time points and endpoints used in this study were similar to those employed in the corresponding adult studies (see Clinical Trials, Invasive Candidiasis, Candidaemia, Oesophageal Candidiasis, Invasive Aspergillosis). All patients received CANCIDAS at 50 mg/m2 IV once daily following a 70-mg/m2 loading dose on Day 1 (not to exceed 70 mg daily). Among the 49 enrolled patients who received CANCIDAS, 48 were included in the MITT analysis. Of these 48 patients, 37 had invasive candidiasis, 10 had invasive aspergillosis, and 1 patient had oesophageal candidiasis. The favourable response rate, by indication, at the end of caspofungin therapy was as follows in the MITT analysis: 81% (30/37) in invasive candidiasis, 50% (5/10) in invasive aspergillosis, and 100% (1/1) in oesophageal candidiasis.

INDICATIONS

CANCIDAS is indicated for:  Empirical therapy for presumed fungal infections in febrile, neutropenic patients whose fever has failed to respond to broad-spectrum antibiotics

 Treatment of: -Invasive Candidiasis, including candidaemia -Oesophageal Candidiasis -Invasive Aspergillosis in patients who are refractory to or intolerant of other therapies.

CONTRAINDICATIONS

CANCIDAS is contraindicated in patients with hypersensitivity to any component of this product.

PRECAUTIONS

General

Anaphylaxis has been reported during administration of CANCIDAS. If this occurs, CANCIDAS should be discontinued and appropriate treatment administered. Possibly histamine-mediated adverse reactions, including rash, facial swelling, angioedema, pruritus, sensation of warmth, or bronchospasm have been reported and may require discontinuation and/or administration of appropriate treatment.

Cases of Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) have been reported after post marketing use of caspofungin. Caution should apply in patients with history of allergic skin reactions.

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Safety information on treatment durations longer than 4 weeks is limited, however available data suggest that CANCIDAS continues to be well tolerated with longer courses of therapy.

Laboratory abnormalities in liver function tests have been seen in healthy volunteers and in adult and paediatric patients treated with CANCIDAS. In some adult and paediatric patients with serious underlying conditions who were receiving multiple concomitant medications with CANCIDAS, isolated cases of clinically significant hepatic dysfunction, hepatitis, and hepatic failure have been reported; a causal relationship to CANCIDAS has not been established. Patients who develop abnormal liver function tests during CANCIDAS therapy should be monitored for evidence of worsening hepatic function and evaluated for risk/benefit of continuing CANCIDAS therapy.

Carcinogenesis / Mutagenesis / Impairment of Fertility

No long-term studies in animals have been performed to evaluate the carcinogenic potential of caspofungin.

Caspofungin did not show evidence of genotoxic potential when evaluated in assays for gene mutation [bacterial (Ames) and mammalian cell (V79 Chinese hamster lung fibroblasts) assays] and chromosomal damage (Chinese hamster ovary cells in vitro and the mouse bone marrow chromosomal assay). Caspofungin was also negative in the alkaline elution/rat hepatocytes DNA strand break test.

Fertility and reproductive performance were not affected by the intravenous administration of caspofungin to rats at IV doses of up to 5 mg/kg/day. At 5 mg/kg/day, drug exposures (AUC) were similar to those seen in patients treated with the 70 mg dose.

Use in Pregnancy (Category B3)

CANCIDAS was shown to be weakly embryotoxic in rats and rabbits. In rats, CANCIDAS was shown to cause the complete ossification of the skull and torso and an increased incidence of cervical rib. Caspofungin also produced increases in resorptions in rats and rabbits and pre-implantation losses in rats. These findings were observed at doses that produced drug exposures similar to those seen in patients treated with a 70 mg dose (1-2 fold clinical exposure at the maximum recommended dose, based on AUC). Caspofungin crossed the placental barrier in rats and rabbits and was detected in the plasma of foetuses of pregnant animals dosed with CANCIDAS. There were no adequate and well-controlled studies in pregnant women. CANCIDAS should be used in pregnancy only if the potential benefit justifies the potential risk to the foetus.

Use in Lactation

Caspofungin was found in the milk of lactating, drug-treated rats. It is not known whether caspofungin is excreted in human milk. Because many drugs are excreted in human milk, women receiving CANCIDAS should not breast-feed.

Use in children

The use of caspofungin in paediatric patients 3 months to 17 years of age is supported by efficacy/safety studies in adults, pharmacokinetic studies in paediatric patients (see Pharmacokinetics subsection) and two prospective efficacy studies in S-WPC-MK0991-IV-102016 14 paediatric patients (see Clinical Trials section).

The efficacy and safety of caspofungin has not been studied in prospective clinical trials involving neonates and infants under 3 months of age.

Caspofungin has not been studied in paediatric patients with endocarditis, osteomyelitis, and meningitis due to Candida. Caspofungin has also not been studied as initial therapy for invasive aspergillosis in paediatric patients.

The pharmacokinetic, efficacy, and safety data for patients aged 3 to 12 months of age are limited. Caution is advised when treating this age group.

Interactions with Other Drugs Table 5 Co-administered drug CANCIDAS N Results dose Cyclosporin A 70 mg daily for 19 Approximately 35% increase in 4 mg/kg as a single dose 11 days CANCIDAS AUC when co- administered with Cyclosporin A*; no effect of CANCIDAS on cyclosporin A Amphotericin B 50 mg daily for 21 No effect of Amphotericin B on 0.25 mg/kg as a single dose 11 days CANCIDAS; no effect of CANCIDAS on Amphotericin B Itraconazole 50 mg daily for 47 No effect of itraconazole on 200mg/d, multiple doses 14 days CANCIDAS; no effect of CANCIDAS on itraconazole Mycophenolate 50 mg daily for 18 No effect of mycophenolate on 1.5g as a single dose 16 days CANCIDAS; no effect of CANCIDAS on mycophenolate Tacrolimus 70 mg or 50 mg 51 No effect of tacrolimus on 0.1 mg/kg, doses on days 1 daily CANCIDAS; 20% decrease in AUC and 10 for tacrolimus when co-administered with CANCIDAS. Rifampicin 600 mg oral dose daily 50 mg daily for 14 Approximately 30 % decrease in When CANCIDAS added 14 days (for both trough concentrations of CANCIDAS, to pre-existing rifampicin studies) but with little change in AUC for (i.e. steady state) CANCIDAS. No effect of therapy CANCIDAS on rifampicin.

When CANCIDAS and 10 Approximately 60% increase in AUC rifampicin initiated on the for CANCIDAS on Day 1. No effect same day of CANCIDAS on rifampicin.

Refer to Dosage and Administration section

Nelfinavir 50 mg daily for 9 No effect of nelfinavir on 1250 mg oral dose twice 14 days CANCIDAS. daily *Refer below for trial description Cyclosporin Concomitant use of CANCIDAS with cyclosporin has been evaluated in healthy adult volunteers and in adult patients. In one clinical study, 3 of 4 healthy adult subjects who received CANCIDAS 70 mg on Days 1 through 10, and also received two 3 mg/kg doses of cyclosporin 12 hours apart on Day 10, developed transient elevations of alanine transaminase (ALT) on Day 11 that were 2 to 3 times the upper limit of S-WPC-MK0991-IV-102016 15 normal (ULN). In a separate panel of adult subjects in the same study, 2 of 8 who received CANCIDAS 35 mg daily for 3 days and cyclosporin (two 3 mg/kg doses administered 12 hours apart) on Day 1 had small increases in ALT (slightly above the ULN) on Day 2. In both groups, elevations in aspartate transaminase (AST) paralleled ALT elevations, but were of lesser magnitude (see ADVERSE REACTIONS, Laboratory Abnormalities).

In the above two clinical studies, cyclosporin increased the AUC of caspofungin by approximately 35%. These AUC increases are probably due to reduced uptake of caspofungin by the liver. CANCIDAS did not increase the plasma levels of cyclosporin. Refer to Table 5.

A retrospective study was conducted of 40 adult patients treated during marketed use with CANCIDAS and cyclosporin for 1 to 290 days (median 17.5 days). The majority of patients had allogeneic haematopoietic stem cell transplants (82.5%) or solid organ transplants (10%). The majority of patients received caspofungin 50 mg daily after 70 mg on Day 1. No serious hepatic adverse events were noted during this study. As expected in this population, hepatic enzyme abnormalities occurred commonly; however, no patient had elevations in ALT that were considered drug related. Elevations in AST considered at least possibly related to therapy with CANCIDAS and/or cyclosporin occurred in 5 patients, but all were less than 3.6 times the ULN. Discontinuations due to laboratory abnormalities in hepatic enzymes from any cause occurred in 4 patients. Of these, 2 were considered possibly related to therapy with CANCIDAS and/or cyclosporin as well as other possible causes. In the prospective invasive aspergillosis and compassionate use studies, there were 6 patients treated with CANCIDAS and cyclosporin for 2 to 56 days; none of these patients experienced increases in hepatic enzymes.

These data suggest that CANCIDAS can be used in patients receiving cyclosporin when the potential benefit outweighs the potential risk.

Tacrolimus Clinical studies in healthy adult volunteers show that the pharmacokinetics of CANCIDAS are not altered by tacrolimus. CANCIDAS reduced the blood AUC of tacrolimus by approximately 20%, maximal blood concentration (Cmax) by 16%, and 12-hour blood concentration (C12hr) by 26% in healthy subjects when tacrolimus (2 doses of 0.1 mg/kg 12 hours apart) was administered on the 10th day of CANCIDAS 70 mg daily, as compared to results from a control period in which tacrolimus was administered alone. For patients receiving both therapies, standard monitoring of tacrolimus blood concentrations and appropriate tacrolimus dosage adjustments are recommended. Refer to Table 5.

Effect of caspofungin on the P450 (CYP) system Studies in vitro show that caspofungin is not an inhibitor of cytochrome P450 (CYP) mediated reactions. In clinical studies, caspofungin did not induce the CYP3A4 metabolism of other drugs. Caspofungin is not a substrate for P-glycoprotein, and metabolism by cytochrome P450 was not observed in vitro.

Other Drugs Clinical studies in healthy adult volunteers show that the pharmacokinetics of CANCIDAS are not altered by itraconazole, amphotericin B, nelfinavir or mycophenolate. CANCIDAS has no effect on the pharmacokinetics of itraconazole, amphotericin B, rifampicin or the active metabolite of mycophenolate. Refer to Table 5.

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Population pharmacokinetic screening of caspofungin concentrations in adult patients receiving other concomitant medications indicate that elevations in plasma caspofungin levels due to drug interactions, as was seen in the formal drug interaction study with cyclosporin, are uncommon. Results from two clinical drug interaction studies indicate that rifampicin both induces and inhibits caspofungin disposition with net induction at steady state. In one study, rifampicin and caspofungin were co-administered for 14 days with both therapies initiated on the same day. In the second study, rifampicin was administered alone for 14 days to allow the induction effect to reach steady state, and then rifampicin and caspofungin were co-administered for an additional 14 days. When the induction effect of rifampicin was at steady state, there was little change in caspofungin AUC or end-of- infusion concentration, but caspofungin trough concentrations were reduced by approximately 30%. The inhibitory effect of rifampicin was demonstrated when rifampicin and caspofungin treatments were initiated on the same day, and a transient elevation in caspofungin plasma concentrations occurred on Day 1 (approximately 60% increase in AUC). This inhibitory effect was not seen when caspofungin was added to pre-existing rifampicin therapy, and no elevation in caspofungin concentrations occurred. Refer to Table 5. In addition, results from the population pharmacokinetic screening suggest that co-administration of other inducers of drug clearance (efavirenz, nevirapine, phenytoin, dexamethasone or carbamazepine) with CANCIDAS may also result in clinically meaningful reductions in caspofungin concentrations. Available data suggest that the inducible drug clearance mechanism involved in caspofungin disposition is likely an uptake transport process, rather than metabolism.

When CANCIDAS is co-administered in adult patients with inducers of drug clearance, such as efavirenz, nevirapine, phenytoin, rifampicin, dexamethasone, or carbamazepine, use of a daily dose of 70 mg CANCIDAS should be considered.

In paediatric patients, results from regression analyses of pharmacokinetic data suggest that co-administration of dexamethasone with caspofungin may result in clinically meaningful reductions in caspofungin trough concentrations. This finding may indicate that paediatric patients will have similar reductions with inducers as seen in adults. When caspofungin is co-administered to paediatric patients with inducers of drug clearance, such as rifampicin, efavirenz, nevirapine, phenytoin, dexamethasone, or carbamazepine, a caspofungin dose of 70-mg/m2 daily (not to exceed an actual daily dose of 70 mg) may need to be considered depending on the clinical response.

ADVERSE EFFECTS

General Hypersensitivity reactions have been reported (see PRECAUTIONS).

Clinical Adverse Experiences in Adult Patients The overall safety of caspofungin was assessed in 1865 adult individuals who received single or multiple doses of caspofungin acetate: 564 febrile, neutropenic patients (empirical therapy study), 382 patients with invasive candidiasis, 297 patients with oesophageal and/or oropharyngeal candidiasis, 228 patients with invasive aspergillosis and 394 individuals in phase I studies. In the empirical therapy study, patients had received chemotherapy for malignancy or had undergone haematopoietic stem-cell transplantation. In the studies involving patients with documented Candida infections, the majority of the patients had serious underlying medical conditions (e.g. haematologic or other malignancy, recent major surgery, S-WPC-MK0991-IV-102016 17

HIV) requiring multiple concomitant medications. Patients in the non-comparative Aspergillus study often had serious predisposing medical conditions (e.g., bone marrow or peripheral stem cell transplants, haematologic malignancy, solid tumours or organ transplants) requiring multiple concomitant medications.

Empirical Therapy in Febrile, Neutropenic Patients In the randomised, double-blinded empirical therapy study, patients received either CANCIDAS 50 mg/day (following a 70 mg loading dose) or AmBisome (3 mg/kg/day). Drug-related clinical adverse experiences occurring in  2% of the patients in either treatment group are presented in Table 6.

Table 6 Drug-Related* Clinical Adverse Experiences Among Patients with Persistent Fever and Neutropenia Incidence  2% for at least one treatment group by Body System CANCIDAS** AmBisome*** N=564 (percent) N=547 (percent) Body as a Whole Abdominal Pain 1.4 2.4 Chills 13.8 24.7 Fever 17.0 19.4 Flushing 1.8 4.2 Perspiration/Diaphoresis 2.8 2.2 Cardiovascular System Hypertension 1.1 2.0 Tachycardia 1.4 2.4 Digestive System Diarrhoea 2.7 2.4 Nausea 3.5 11.3 Vomiting 3.5 8.6 Metabolism and Nutrition Hypokalaemia 3.7 4.2 Musculoskeletal System Back Pain 0.7 2.7 Nervous System & Psychiatric Headache 4.3 5.7 Respiratory System Dyspnoea 2.0 4.2 Tachypnoea 0.4 2.0 Skin & Skin Appendage Rash 6.2 5.3 * Determined by the investigator to be possibly, probably, or definitely drug-related. ** 70 mg on Day 1, then 50 mg daily for the remainder of treatment; daily dose was increased to 70 mg for 73 patients. *** 3.0 mg/kg/day; daily dose was increased to 5.0 mg/kg for 74 patients.

The proportion of patients who experienced an infusion-related adverse event was significantly lower in the group treated with CANCIDAS (35.1%) than in the group treated with AmBisome (51.6%).

To evaluate the effect of CANCIDAS and AmBisome on renal function, nephrotoxicity was defined as doubling of serum creatinine relative to baseline or an increase of 1 mg/dL in serum creatinine if baseline serum creatinine was above the upper limit of the normal range. Among patients whose baseline creatinine clearance was >30 mL/min, the incidence of nephrotoxicity was significantly lower in the group treated with CANCIDAS (2.6%) than in the group treated with AmBisome (11.5%)

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Invasive Candidiasis In an initial randomised, double-blinded invasive candidiasis study, patients received either CANCIDAS 50 mg/day (following a 70-mg loading dose) or amphotericin B, 0.6 to 1.0 mg/kg/day. Drug-related clinical adverse experiences occurring in 2% of the patients in either treatment group are presented in Table 7.

Table 7 Drug-Related Clinical Adverse Experiences Among Patients with Invasive Candidiasis* Incidence 2% for at least one treatment group by Body System

CANCIDAS 50 mg** Amphotericin B N=114 (percent) N=125 (percent) Body as a Whole Chills 5.3 26.4 Fever 7.0 23.2 Cardiovascular System Hypertension 1.8 6.4 Hypotension 0.9 2.4 Tachycardia 1.8 10.4 Peripheral Vascular System Phlebitis/thrombophlebitis 3.5 4.8 Digestive System Diarrhoea 2.6 0.8 Jaundice 0.9 3.2 Nausea 1.8 5.6 Vomiting 3.5 8.0

Metabolic/Nutritional/Immune Hypokalaemia 0.9 5.6 Nervous System & Psychiatric Tremor 1.8 2.4 Respiratory System Tachypnoea 0.0 10.4 Skin & Skin Appendage Erythema 0.0 2.4 Rash 0.9 3.2 Sweating 0.9 3.2 Urogenital System Renal insufficiency 0.9 5.6 Renal insufficiency, acute 0.0 5.6 * Determined by the investigator to be possibly, probably, or definitely drug related. ** Patients received CANCIDAS 70 mg on Day 1, then 50 mg daily for the remainder of their treatment.

The incidence of drug-related clinical adverse experiences was significantly lower among patients treated with CANCIDAS (28.9%) than among patients treated with amphotericin B (58.4%). Also, the proportion of patients who experienced an infusion-related adverse event was significantly lower in the CANCIDAS group (20.2%) than in the amphotericin B group (48.8%).

In a second randomised, double-blinded invasive candidiasis study, patients received either CANCIDAS 50 mg/day (following a 70-mg loading dose) or CANCIDAS 150 mg/day. The primary endpoint for this study was the proportion of patients developing a significant drug-related adverse experience (defined as a serious drug-related adverse experience or a drug-related adverse experience leading to caspofungin discontinuation). The proportion of patients with a significant drug-related adverse experience was comparable in the 2 treatment groups: 1.9% (2/104) vs. 3.0% (3/100) in the CANCIDAS 50 mg/day and 150 mg/day groups, respectively (difference 1.1% [95% CI -4.1, 6.8]). The proportion of patients who experienced any drug-related adverse experience was also similar in the 2 treatment groups. Drug-related clinical S-WPC-MK0991-IV-102016 19 adverse experiences occurring in 2.0% of the patients in either treatment group are presented in Table 8.

TABLE 8 Drug-Related* Clinical Adverse Experiences among Patients with Invasive Candidiasis Incidence ≥2.0% for at Least One Treatment Group by System Organ Class or Preferred Term

Adverse Experience CANCIDAS 50 mg† CANCIDAS 150 mg (MedDRA v11.0 System Organ Class and N=104 (percent) N=100 (percent) Preferred Term) All Systems, Any Adverse Experience 13.5 14.0 General Disorders and Administration 6.7 7.0 Site Conditions Injection site erythema 0.0 2.0 Injection site phlebitis 3.8 2.0 Injection site swelling 1.0 2.0 Metabolism and Nutrition Disorders 1.9 2.0 Hypokalemia 1.0 2.0 Nervous System Disorders 0.0 2.0 Headache 0.0 2.0 * Determined by the investigator to be possibly, probably, or definitely drug-related. Within any system organ class, individuals may experience more than 1 adverse experience † Patients received CANCIDAS 70 mg on Day 1, then 50 mg daily for the remainder of their treatment

Oesophageal and/or oropharyngeal candidiasis Drug-related clinical adverse experiences occurring in 2% of patients with oesophageal and/or oropharyngeal candidiasis are presented in Table 9. S-WPC-MK0991-IV-102016 20

TABLE 9 Drug-related Clinical Adverse Experiences among Patients with Oesophageal and/or Oropharyngeal Candidiasis* Incidence 2% for at least one treatment dose (per comparison) by Body System

CANCIDAS Fluconazole CANCIDAS CANCIDAS Amphotericin B 50 mg IV 200mg 50 mg 70 mg 0.5 mg/kg (N=83) (N=94) (N=80) (N=65) (N=89) percent percent percent percent percent Body as a Whole Asthenia/fatigue 0.0 0.0 0.0 0.0 6.7 Chills 0.0 0.0 2.5 1.5 75.3 Oedema/swelling 0.0 0.0 0.0 0.0 5.6 Oedema, facial 0.0 0.0 0.0 3.1 0.0 Fever 3.6 1.1 21.3 26.2 69.7 Flu-like illness 0.0 0.0 0.0 3.1 0.0 Malaise 0.0 0.0 0.0 0.0 5.6 Pain 0.0 0.0 1.3 4.6 5.6 Pain, abdominal 3.6 2.1 2.5 0.0 9.0 Warm sensation 0.0 0.0 0.0 1.5 4.5 Peripheral Vascular System Infused vein complication 12.0 8.5 2.5 1.5 0.0 Phlebitis/thrombophlebitis 15.7 8.5 11.3 13.8 22.5 Cardiovascular System Tachycardia 0.0 0.0 1.3 0.0 4.5 Vasculitis 0.0 0.0 0.0 0.0 3.4 Digestive System Anorexia 0.0 0.0 1.3 0.0 3.4 Diarrhoea 3.6 2.1 1.3 3.1 11.2 Gastritis 0.0 2.1 0.0 0.0 0.0 Nausea 6.0 6.4 2.5 3.1 21.3 Vomiting 1.2 3.2 1.3 3.1 13.5 Haemic & Lymphatic System Anaemia 0.0 0.0 3.8 0.0 9.0 Musculoskeletal System Myalgia 1.2 0.0 0.0 3.1 2.2 Pain, back 0.0 0.0 0.0 0.0 2.2 Pain, musculoskeletal 0.0 0.0 1.3 0.0 4.5 Metabolic/Nutritional /Immune Anaphylaxis 0.0 0.0 0.0 0.0 2.2 Nervous System & Psychiatric Dizziness 0.0 2.1 0.0 1.5 1.1 Headache 6.0 1.1 11.3 7.7 19.1 Insomnia 1.2 0.0 0.0 0.0 2.2 Paraesthesia 0.0 0.0 1.3 3.1 1.1 Tremor 0.0 0.0 0.0 0.0 7.9 Respiratory System Tachypnoea 0.0 0.0 1.3 0.0 4.5 Skin & Skin Appendage Erythema 1.2 0.0 1.3 1.5 7.9 Induration 0.0 0.0 0.0 3.1 6.7 Pruritus 1.2 0.0 2.5 1.5 0.0 Rash 0.0 0.0 1.3 4.6 3.4 Sweating 0.0 0.0 1.3 0.0 3.4 *Relationship to drug was determined by the investigator to be possibly, probably or definitely drug-related. Patients who received CANCIDAS 35 mg daily in these studies are not included in this table.

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Invasive aspergillosis In the open-label, non-comparative aspergillosis study, in which 69 patients received CANCIDAS (70-mg loading dose on Day 1 followed by 50 mg daily), the following drug-related clinical adverse experiences were observed with an incidence of 2%: fever (2.9%), infused-vein complications (2.9%), nausea (2.9%), vomiting (2.9%) and flushing (2.9%). Also reported infrequently in this patient population were pulmonary oedema, adult respiratory distress syndrome (ARDS), and radiographic infiltrates.

Clinical Adverse Experiences in Paediatric Patients The overall safety of caspofungin was assessed in 171 paediatric patients who received single or multiple doses of CANCIDAS: 104 febrile, neutropenic patients; 56 patients with invasive candidiasis; 1 patient with oesophageal candidiasis; and 10 patients with invasive aspergillosis. The overall clinical safety profile of CANCIDAS in paediatric patients is comparable to that in adult patients. Table 10 shows the incidence of drug-related clinical adverse experiences reported in 2.0% of paediatric patients in clinical studies. The most common drug-related clinical adverse experiences in paediatric patients treated with CANCIDAS were fever (11.7%), rash (4.7%), and headache (2.9%).

TABLE 10 Drug-Related Clinical Adverse Experiences among Paediatric Patients* Incidence 2% for at least one treatment dose by Body System

CANCIDAS CANCIDAS AmBisome 2 Any Dose** 50 mg/m *** 3 mg/kg*** N=171 N=56 N=26 (percent) (percent) (percent) Cardiac Disorders Tachycardia 1.2 1.8 11.5 Gastrointestinal Disorders Nausea 0.0 0.0 3.8 Vomiting 0.6 1.8 7.7 General Disorders & Administration Site Conditions Adverse Drug Reaction 0.0 0.0 3.8 Catheter Site Pain 1.2 3.6 0.0 Chills 1.8 1.8 7.7 Fever 11.7 28.6 23.1 Hepatobiliary Disorders Hepatitis, toxic 0.0 0.0 3.8 Hyperbilirubinemia 0.0 0.0 3.8 Jaundice 0.0 0.0 3.8 Metabolism & Nutrition Disorders Hypokalemia 0.6 0.0 3.8 Nervous System Disorders Headache 2.9 8.9 0.0 Respiratory, Thoracic, & Mediastinal Disorders Dyspnea 0.0 0.0 3.8 Laryngospasm 0.0 0.0 3.8 Skin & Subcutaneous Tissue Disorders Angioneurotic edema 0.0 0.0 3.8 Circumoral edema 0.0 0.0 3.8 Pruritus 1.8 3.6 0.0 Rash 4.7 8.9 0.0 Vascular Disorders Flushing 1.8 3.6 0.0 Hypotension 1.8 3.6 3.8 * Relationship to drug was determined by the investigator to be possibly, probably or definitely drug-related. ** Derived from all paediatric clinical studies. ***Derived from Phase II comparator-controlled clinical study of empirical therapy.

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One patient (0.6%) receiving CANCIDAS and three patients (11.5%) receiving AmBisome developed a serious drug-related clinical adverse experience. Two patients (1.2%) were discontinued from CANCIDAS and three patients (11.5%) were discontinued from AmBisome due to a drug-related clinical adverse experience. The proportion of patients who experienced an infusion-related adverse event was 21.6% in the group treated with CANCIDAS and 34.6% in the group treated with AmBisome.

Laboratory values: Adult Patients Empirical Therapy in Febrile, Neutropenic Patients

Drug-related laboratory adverse experiences occurring in  2% of the patients in either treatment group are presented in Table 11.

TABLE 11 Drug-Related* Laboratory Adverse Experiences among Patients with Persistent Fever and Neutropenia Incidence  2% for at least one treatment group by Laboratory Test Category CANCIDAS** AmBisome*** N=564 (percent) N=547 (percent) Blood Chemistry Alanine aminotransferase increased 8.7 8.9 Alkaline phosphatase increased 7.0 12.0 Aspartate aminotransferase increased 7.0 7.6 Direct serum bilirubin increased 2.6 5.2 Total serum bilirubin increased 3.0 5.2 Hypokalemia 7.3 11.8 Hypomagnesemia 2.3 2.6 Serum creatinine increased 1.2 5.5 * Determined by the investigator to be possibly, probably, or definitely drug-related. ** 70 mg on Day 1, then 50 mg daily for the remainder of treatment; daily dose was increased to 70 mg for 73 patients. *** 3.0 mg/kg/day; daily dose was increased to 5.0 mg/kg for 74 patients.

The percentage of patients with either a drug-related clinical or a drug-related laboratory adverse experience was significantly lower among patients receiving CANCIDAS (54.4%) than among patients receiving AmBisome (69.3%). Furthermore, the incidence of discontinuation due to a drug-related clinical or laboratory adverse experience was significantly lower among patients treated with CANCIDAS (5.0%) than among patients treated with AmBisome (8.0%).

Invasive Candidiasis Drug-related laboratory adverse experiences occurring in 2% of the patients in an initial invasive candidiasis study are presented in Table 12.

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Table 12 Drug-Related Laboratory Adverse Experiences among Patients with Invasive Candidiasis* Incidence 2% for at least one treatment group by Laboratory Test Category

CANCIDAS 50 mg** Amphotericin B N=114 (percent) N=125 (percent) Blood Chemistry ALT increased 3.7 8.1 AST increased 1.9 9.0 Blood urea increased 1.9 15.8 Direct serum bilirubin increased 3.8 8.4 Serum alkaline phosphatase increased 8.3 15.6 Serum bicarbonate decreased 0.0 3.6 Serum creatinine increased 3.7 22.6 Serum phosphate increased 0.0 2.7 Serum potassium decreased 9.9 23.4 Serum potassium increased 0.9 2.4 Total serum bilirubin increased 2.8 8.9 Haematology Haematocrit decreased 0.9 7.3 Haemoglobin decreased 0.9 10.5 Urinalysis Urine protein increased 0.0 3.7 * Determined by the investigator to be possibly, probably, or definitely drug related. ** Patients received CANCIDAS 70 mg on Day 1, then 50 mg daily for the remainder of their treatment.

The incidence of drug-related laboratory adverse experiences was significantly lower among patients receiving CANCIDAS (24.3%) than among patients receiving amphotericin B (54.0%).

The percentage of patients with either a drug-related clinical adverse experience or a drug-related laboratory adverse experience was significantly lower among patients receiving CANCIDAS (42.1%) than among patients receiving amphotericin B (75.2%). Furthermore, a significant difference between the two treatment groups was observed with regard to incidence of discontinuation due to drug-related clinical or laboratory adverse experience; 3/114 (2.6%) in the CANCIDAS group and 29/125 (23.2%) in the amphotericin B group.

To evaluate the effect of CANCIDAS and amphotericin B on renal function, nephrotoxicity was defined as doubling of serum creatinine relative to baseline or an increase of 1 mg/dL in serum creatinine if baseline serum creatinine was above the upper limit of the normal range. In a subgroup of patients whose baseline creatinine clearance was >30 mL/min, the incidence of nephrotoxicity was significantly lower in the CANCIDAS group than in the amphotericin B group.

In a second randomised, double-blinded invasive candidiasis study, patients received either CANCIDAS 50 mg/day (following a 70-mg loading dose) or CANCIDAS 150 mg/day. Drug-related laboratory adverse experiences occurring in 2.0% of the patients in either treatment group are presented in Table 13. S-WPC-MK0991-IV-102016 24

TABLE 13 Drug-Related* Laboratory Adverse Experiences among Patients with Invasive Candidiasis Incidence ≥2.0% for at Least One Treatment Group by System Organ Class or Preferred Term

Adverse Experience CANCIDAS 50 mg† CANCIDAS 150 mg (MedDRA v11.0 System Organ Class and N=104 (percent) N=100 (percent) Preferred Term) All Systems, Any Adverse Experience 7.8 7.1 Alanine Aminotransferase Increased 2.0 2.0 Alkaline Phosphatase Increased 6.9 2.0 Aspartate Aminotransferase Increased 4.0 2.0 * Determined by the investigator to be possibly, probably, or definitely drug-related. Within any system organ class, individuals may experience more than 1 adverse experience † Patients received CANCIDAS 70 mg on Day 1, then 50 mg daily for the remainder of their treatment

Oesophageal and/or oropharyngeal candidiasis Drug-related laboratory abnormalities occurring in 2% of patients with oesophageal and/or oropharyngeal candidiasis are presented in Table 14.

TABLE 14 Drug-related Laboratory Abnormalities Reported among Patients with Oesophageal and/or Oropharyngeal Candidiasis (comparative studies)* Incidence 2% (for at least one treatment dose) by Laboratory Test Category CANCIDAS CANCIDAS Fluconazole Amphotericin 50 mg 70 mg 200 mg B 0.5 mg/Kg (N=163 ) (N=65 ) (N=94 ) (N=89 ) (percent) (percent) (percent) (percent) Blood Chemistry ALT increased 10.6 10.8 11.8 22.7 AST increased 13.0 10.8 12.9 22.7 Blood urea increased 0.0 0.0 1.2 10.3 Direct serum bilirubin increased 0.6 0.0 3.3 2.5 Serum albumin decreased 8.6 4.6 5.4 14.9 Serum alkaline phosphatase increased 10.5 7.7 11.8 19.3 Serum bicarbonate decreased 0.9 0.0 0.0 6.6 Serum calcium decreased 1.9 0.0 3.2 1.1 Serum creatinine increased 0.0 1.5 2.2 28.1 Serum potassium decreased 3.7 10.8 4.3 31.5 Serum potassium increased 0.6 0.0 2.2 1.1 Serum sodium decreased 1.9 1.5 3.2 1.1 Serum uric acid increased 0.6 0.0 0.0 3.4 Total serum bilirubin increased 0.0 0.0 3.2 4.5 Total serum protein decreased 3.1 0.0 3.2 3.4 Haematology Eosinophils increased 3.1 3.1 1.1 1.1 Haematocrit decreased 11.1 1.5 5.4 32.6 Haemoglobin decreased 12.3 3.1 5.4 37.1 Lymphocytes increased 0.0 1.6 2.2 0.0 Neutrophils decreased 1.9 3.1 3.2 1.1 Platelet count decreased 3.1 1.5 2.2 3.4 Prothrombin time increased 1.3 1.5 0.0 2.3 WBC count decreased 6.2 4.6 8.6 7.9 Urinalysis Urine blood increased 0.0 0.0 0.0 4.0 Urine casts increased 0.0 0.0 0.0 8.0 Urine pH increased 0.8 0.0 0.0 3.6 Urine protein increased 1.2 0.0 3.3 4.5 Urine RBC's increased 1.1 3.8 5.1 12.0 Urine WBC's increased 0.0 7.7 0.0 24.0 S-WPC-MK0991-IV-102016 25

* Relationship to drug was determined by the investigator to be possibly, probably, or definitely drug-related. Patients who received CANCIDAS 35 mg daily in these studies are not included in this table.

Invasive aspergillosis Drug-related laboratory abnormalities reported with an incidence 2% in patients treated with CANCIDAS in the non-comparative aspergillosis study were: serum alkaline phosphatase increased (2.9%), serum potassium decreased (2.9%), eosinophils increased (3.2%), urine protein increased (4.9%), and urine RBCs increased (2.2%).

The safety and efficacy of multiple doses up to 150 mg daily (range: 1 to 51 days; median: 14 days) have been studied in 100 adult patients with invasive candidiasis. CANCIDAS was generally well tolerated in these patients receiving CANCIDAS at this higher dose; however, the efficacy of CANCIDAS at this higher dose was generally similar to patients receiving the 50-mg daily dose of CANCIDAS.

Paediatric Patients Table 15 shows the incidence of drug-related laboratory adverse experiences reported in 2.0% of paediatric patients in clinical studies. The overall laboratory safety profile in paediatric patients is comparable to that in adult patients. The most common drug-related laboratory adverse experiences in paediatric patients treated with CANCIDAS were increased ALT (6.5%) and increased AST (7.6%). None of the patients receiving CANCIDAS or AmBisome developed a serious drug-related adverse event or were discontinued from therapy due to a drug-related laboratory adverse experience.

TABLE 15 Drug-Related Laboratory Adverse Experiences among Paediatric Patients* Incidence 2% for at least one treatment dose by Body System

CANCIDAS CANCIDAS AmBisome Any Dose** 50 mg/m2*** 3 mg/kg*** N=171 N=56 N=26 (percent) (percent) (percent) Blood Chemistry Test Alanine aminotransferase (ALT) increased 6.5 3.6 0.0 Aspartate aminotransferase (AST) 7.6 1.8 0.0 increased Blood bilirubin increased 0.6 1.8 4.0 Blood phosphorus decreased 2.0 1.8 0.0 Blood potassium decreased 3.5 3.6 11.5 Blood sodium decreased 0.0 0.0 3.8 Direct bilirubin increased 0.0 0.0 6.3 * Relationship to drug was determined by the investigator to be possibly, probably or definitely drug-related. **Derived from all paediatric clinical studies. ***Derived from Phase II comparator-controlled clinical study of empirical therapy.

Post-marketing experience The following post-marketing adverse events have been reported:

Hepatobiliary: rare cases of hepatic dysfunction Skin and subcutaneous tissue disorders: toxic epidermal necrolysis and Steven- Johnson syndrome Cardiovascular: swelling and peripheral oedema Metabolic: hypercalcaemia; gamma-glutamyltransferase increased.

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DOSAGE AND ADMINISTRATION

Dosage Recommendations

CANCIDAS should be administered in adult patients by slow intravenous infusion over approximately 1 hour (refer to Reconstitution section for dilution recommendations).

Table 16 Dosing Recommendations in Adult Patients

Loading Dose Maintenance dose Empirical Therapy Normal hepatic function or 70 mg on day 1 50 mg daily mild hepatic insufficiency* Moderate hepatic 70 mg on day 1 35 mg daily insufficiency **

Invasive candidiasis Normal hepatic function or 70 mg on day 1 50 mg daily mild hepatic insufficiency* Moderate hepatic 70 mg on day 1 35 mg daily insufficiency **

Oesophageal Normal hepatic function or Not required 50 mg daily mild hepatic insufficiency* Moderate hepatic Not required 35 mg daily insufficiency**

Invasive aspergillosis Normal hepatic function or 70 mg on day 1 50 mg daily mild hepatic insufficiency* Moderate hepatic 70 mg on day 1 35 mg daily insufficiency ** *Mild hepatic insufficiency: Child-Pugh score 5 to 6 **Moderate hepatic insufficiency: Child Pugh score 7 to 9

General Recommendations in Adult Patients

Empirical Therapy Duration of treatment should be based on the patient’s clinical response. Empirical therapy should be continued until resolution of neutropenia, which is generally expected to occur within 28 days. Patients found to have a fungal infection should be treated for a minimum of 14 days; treatment should continue for at least 7 days after both neutropenia and clinical symptoms are resolved. If the 50-mg dose is well tolerated but does not provide an adequate clinical response, the daily dose can be increased to 70 mg. Although an increase in efficacy with 70 mg daily has not been demonstrated, safety data suggest that an increase in dose to 70 mg daily is well tolerated.

Candidiasis  Invasive: Duration of treatment of invasive candidiasis should be dictated by the patient’s clinical and microbiological response. In general, antifungal therapy S-WPC-MK0991-IV-102016 27

should continue for at least 14 days after the last positive culture. Patients who remain persistently neutropenic may warrant a longer course of therapy pending resolution of the neutropenia.  Oesophageal: Increasing doses of CANCIDAS above 50 mg daily provided no additional benefit in the treatment of oesophageal candidiasis.

Invasive Aspergillosis Duration of treatment should be based upon the severity of the patient’s underlying disease, recovery from immunosuppression, and clinical response. The efficacy of a 70-mg dose regimen in patients who are not clinically responding to the 50-mg daily dose is not known. Safety data suggest that an increase in dose to 70 mg daily is well tolerated. The efficacy of doses above 70 mg has not been adequately studied in patients with invasive aspergillosis.

Concomitant Therapy with Inducers of Drug Clearance When CANCIDAS is co-administered in adult patients with inducers of drug clearance, such as efavirenz, nevirapine, phenytoin, rifampicin, dexamethasone or carbamazepine, use of a daily dose of 70 mg of CANCIDAS should be considered.

Hepatic Insufficiency  Adult patients with mild hepatic insufficiency (Child-Pugh score 5 to 6) do not need a dosage adjustment.  Adult patients with moderate hepatic insufficiency (Child-Pugh score 7 to 9), require an adjustment to maintenance dosage-refer to Table16.  There is no clinical experience in adult patients with severe hepatic insufficiency (Child-Pugh score >9) (see PHARMACOLOGY, Pharmacokinetics, Special Populations) and in paediatric patients with any degree of hepatic insufficiency.

Renal Insufficiency No dosage adjustment is necessary for patients with renal insufficiency. Caspofungin is not dialysable, thus supplementary dosing is not required following haemodialysis (see PHARMACOLOGY, Pharmacokinetics, Special Populations).

Paediatric Patients CANCIDAS should be administered in paediatric patients (3 months to 17 years of age) by slow IV infusion over approximately 1 hour. Dosing in paediatric patients (3 months to 17 years of age) should be based on the patient’s body surface area (see Instructions for Use in Paediatric Patients, Mosteller2 Formula).

For all indications, a single 70-mg/m2 loading dose (not to exceed an actual dose of 70 mg) should be administered on Day 1, followed by 50 mg/m2 daily thereafter (not to exceed an actual dose of 70 mg daily). Duration of treatment should be individualised to the indication, as described for each indication in adults (see General Recommendations in Adult Patients).

If the 50-mg/m2 daily dose is well tolerated but does not provide an adequate clinical response, the daily dose can be increased to 70 mg/m2 daily (not to exceed an actual daily dose of 70 mg). Although an increase in efficacy with 70 mg/m2 daily has not been demonstrated, limited safety data suggest that an increase in dose to 70 mg/m2 daily is well tolerated.

The efficacy and safety of CANCIDAS have not been sufficiently studied in clinical trials involving neonates and infants below 12 months of age. Caution is advised

2 Mosteller RD: Simplified Calculation of Body Surface Area. N Engl J Med 1987 Oct 22;317(17): 1098 (letter)

S-WPC-MK0991-IV-102016 28 when treating this age group. Limited data suggest that CANCIDAS at 25 mg/m2 daily in neonates and infants (less than 3 months of age) and 50 mg/m2 daily in young children (3 to 11 months of age) can be considered.

When CANCIDAS is co-administered to paediatric patients with inducers of drug clearance, such as rifampicin, efavirenz, nevirapine, phenytoin, dexamethasone, or carbamazepine, use of a CANCIDAS dose of 70-mg/m2 daily (not to exceed an actual daily dose of 70 mg) may need to be considered depending on the clinical response.

Reconstitution of CANCIDAS

The reconstituted vial of CANCIDAS must be further diluted prior to administration. DO NOT USE DILUENTS CONTAINING GLUCOSE as CANCIDAS is not stable in diluents containing glucose.

CANCIDAS should be administered by slow IV infusion of approximately 1 hour. It contains no antimicrobial agent. Product is for single use in one patient only. Discard any residue.

Do not mix or co-infuse CANCIDAS with other medications as there is no data available on the compatibility of CANCIDAS with other intravenous substances, additives, or medications.

INSTRUCTIONS FOR USE IN ADULTS

Step 1: Reconstitution of vials To reconstitute the powdered drug, bring the refrigerated vial of CANCIDAS to room temperature and aseptically add 10.5 mL of 0.9% Sodium Chloride Injection or Water for Injections. The concentrations of the reconstituted vials will be: 7.2 mg/mL (70 mg vial) or 5.2 mg/mL (50 mg vial).

The white to off-white compact powder will dissolve completely. Mix gently until a clear solution is obtained. Reconstituted solutions should be visually inspected for particulate matter or discolouration. This reconstituted solution is chemically and physically stable for up to 1 hour when held below 25°C. However, to reduce microbiological hazard, use as soon as practicable after dilution and if storage is necessary, hold at 2-8C for not more than 1 hour.

Step 2: Addition of reconstituted CANCIDAS to infusion solution The patient infusion solution is prepared by aseptically adding the appropriate amount of reconstituted drug (as shown in Table17) to a 250 mL intravenous PVC bag or glass bottle of 0.9% Sodium Chloride Injection. Reduced volume infusions in 100 mL may be used, when medically necessary, for 50 mg or 35 mg daily doses.

Visually inspect the infusion solution for particulate matter or discolouration. Do not use if the solution is cloudy or precipitated. This infusion solution is chemically and physically stable for 24 hours when stored below 25C. However, to reduce microbiological hazard, use as soon as practicable after dilution and if storage is necessary, hold at 2-8C for not more than 24 hours. CANCIDAS should be administered by slow intravenous infusion over approximately 1 hour. It contains no antimicrobial agent. Use once only and discard any residue. S-WPC-MK0991-IV-102016 29

Table 17: Preparation of the patient infusion solutions

Volume of Typical Preparation Reduced Volume reconstituted (reconstituted Infusion DOSE* CANCIDAS for CANCIDAS added to (reconstituted CANCIDAS transfer to 250 mL infusion added to 100mL infusion intravenous bag sodium chloride 0.9%) sodium chloride 0.9% or bottle final concentration final concentration 70 mg (from one 70 10 mL 0.28 mg/mL not recommended mg vial) 70 mg (from two 50 14 mL 0.28 mg/mL not recommended mg vials)** 50 mg (from one 50 10 mL 0.20 mg/mL 0.47 mg/mL mg vial) 35 mg (from one 50 7 mL 0.14 mg/mL 0.34 mg/mL mg vial) 35 mg (from one 70 5 mL 0.14 mg/mL 0.34 mg/mL mg vial) * 10.5 mL should be used for reconstitution of all vials **If 70 mg vial is not available, the 70 mg dose can be prepared from two 50 mg vials

INSTRUCTIONS FOR USE IN PAEDIATRIC PATIENTS

Calculation of Body Surface Area (BSA) for paediatric dosing Before preparation of infusion, calculate the body surface area (BSA) of the patient using the following formula: (Mosteller Formula)

Preparation of the 70 mg/m2 infusion for paediatric patients 3 months of age or older (using a 70-mg vial) 1. Determine the actual loading dose to be used in the paediatric patient by using the patient's BSA (as calculated above) and the following equation: BSA (m2) X 70 mg / m2 = Loading Dose The maximum loading dose on Day 1 should not exceed 70 mg regardless of the patient's calculated dose. 2. Equilibrate the refrigerated vial of CANCIDAS to room temperature. 3. Aseptically add 10.5 mL of 0.9% Sodium Chloride Injection or Sterile Water for Injectiona This reconstituted solution may be stored for up to one hour at ≤25°C.b This will give a final caspofungin concentration in the vial of 7.2 mg/mL. 4. Remove the volume of drug equal to the calculated loading dose (Step 1) from the vial. Aseptically transfer this volume (mL)c of reconstituted CANCIDAS to an IV bag (or bottle) containing 250 mL of 0.9%, 0.45%, or 0.225% Sodium Chloride Injection, or Lactated Ringers Injection. Alternatively, the volume (mL)c of reconstituted CANCIDAS can be added to a reduced volume of 0.9%, 0.45%, or 0.225% Sodium Chloride Injection or Lactated Ringers Injection, not to exceed a S-WPC-MK0991-IV-102016 30

final concentration of 0.5 mg/mL. This infusion solution must be used within 24 hours if stored at ≤25°C or within 24 hours if stored refrigerated at 2 to 8°C. 5. If the calculated loading dose is <50 mg, then the dose may be prepared from the 50-mg vial [follow Steps 2-4 from Preparation of the 50 mg/m2 infusion for paediatric patients 3 months of age or older (using a 50-mg vial)]. The final caspofungin concentration in the 50-mg vial after reconstitution is 5.2 mg/mL.

Preparation of the 50 mg/m2 infusion for paediatric patients 3 months of age or older (using a 50-mg vial) 1. Determine the daily maintenance dose to be used in the paediatric patient by using the patient's BSA (as calculated above) and the following equation: BSA (m2) X 50 mg/m2 = Daily Maintenance Dose (mg) The daily maintenance dose should not exceed 70 mg regardless of the patient's calculated dose. 2. Equilibrate the refrigerated vial of CANCIDAS to room temperature. 3. Aseptically add 10.5 mL of 0.9% Sodium Chloride Injection or Sterile Water for Injectiona This reconstituted solution may be stored for up to one hour at ≤25°C.b This will give a final caspofungin concentration in the vial of 5.2 mg/mL. 4. Remove the volume of drug equal to the calculated loading dose (Step 1) from the vial. Aseptically transfer this volume (mL)c of reconstituted CANCIDAS to an IV bag (or bottle) containing 250 mL of 0.9%, 0.45%, or 0.225% Sodium Chloride Injection, or Lactated Ringers Injection. Alternatively, the volume (mL)c of reconstituted CANCIDAS can be added to a reduced volume of 0.9%, 0.45%, or 0.225% Sodium Chloride Injection or Lactated Ringers Injection, not to exceed a final concentration of 0.5 mg/mL. This infusion solution must be used within 24 hours if stored at ≤25°C or within 24 hours if stored refrigerated at 2 to 8°C. 5. If the actual daily maintenance dose is > 50 mg, then the dose may be prepared from the 70-mg vial [follow steps 2-4 from Preparation of the 70 mg/m2 infusion for paediatric patients 3 months of age or older (using a 70-mg vial)]. The final caspofungin concentration in the 70-mg vial after reconstitution is 7.2 mg/L.

Preparation notes: a. The white to off-white cake will dissolve completely. Mix gently until a clear solution is obtained. b. Visually inspect the reconstituted solution for particulate matter or discolouration during reconstitution and prior to infusion. Do not use if the solution is cloudy or has precipitated. c. CANCIDAS is formulated to provide the full labelled vial dose (70 mg or 50 mg) when 10 mL is withdrawn from the vial.

OVERDOSAGE In clinical studies the highest dose was 210 mg, which was administered as a single dose to 6 healthy subjects and was generally well tolerated. In addition, 150 mg daily up to 51 days has been administered to 100 healthy patients and was generally well- tolerated. Caspofungin is not dialysable. S-WPC-MK0991-IV-102016 31

PRESENTATION AND STORAGE CONDITIONS

CANCIDAS is available as a white to off white lyophilised compact powder for infusion in single use vials of 50 mg and 70 mg.

The lyophilised vials of CANCIDAS should be stored at 2-8C, refrigerate, do not freeze.

Reconstituted vials of CANCIDAS may be stored for one hour prior to preparation of the infusion solution, and the final patient infusion solution in the IV bag or bottle can be stored for up to 24 hours. To reduce microbiological hazard, it is recommended to use as soon as practicable after reconstitution/dilution and if storage is necessary, to hold at 2-8C.

SPONSOR

MERCK SHARP & DOHME (AUSTRALIA) PTY LIMITED LEVEL 1, BUILDING A, 26 TALAVERA ROAD MACQUARIE PARK NSW 2113

POISONS SCHEDULE

Schedule 4 – Prescription Medicine

This document was approved by the Therapeutic Goods Administration on 4 November 2010. Date of most recent amendment: 9 February 2017