EXPERT OPINION ON INVESTIGATIONAL DRUGS https://doi.org/10.1080/13543784.2019.1581763

REVIEW Investigational drug therapies currently in early-stage clinical development for the treatment of clostridioides (clostridium) difficile infection Mai-Chi N. Trana,b, Ravina Kullarc and Ellie J. C. Goldsteind,e aDepartment of Pharmacy, Providence St. John’s Health Center, Santa Monica, CA, USA; bDepartment of Pharmacy, Clinica Juan Pablo Medical Group, Los Angeles, CA, USA; cDoctor Evidence, LLC, Santa Monica, CA, USA; dR M Alden Research Laboratory, Santa Monica, CA, USA; eDavid Geffen School of Medicine, Los Angeles, CA, USA

ABSTRACT ARTICLE HISTORY Introduction: Clostridioides (Clostridium) difficile Infection (CDI) is an urgent global threat causing Received 16 August 2018 ~500,000 infections annually in the United States of America (USA) and is associated with a 36% 30- Accepted 8 February 2019 day attributable mortality rate. Despite the availability of three therapeutic agents, CDI recurrence KEYWORDS – – occurs in 20 40% of patients, with a 30 40% second recurrence rate in these patients. Consequently, ACX-362F; DS-2969b; there is a need for novel agents for treating CDI. LFF571; ribaxamase; Areas covered: We searched MEDLINE, PubMed, Embase, Web of Science, Cochrane Central Register of ridinilazole; RBX 2660; Controlled Trials, and ClinicalTrials.gov for agents in early stages of clinical development. CRS3123; MCB3681/ These drugs include ACX-362E, DS-2969b, LFF 571, RBX2660, ribaxamase, ridinilazole that have MCB3837 advanced to at least phase 2 and several other drugs in phase 1 development. Expert opinion: The challenge for these new agents is three-fold: (1) to have a novel approach such as a different target/mechanism of action; (2) be ‘significantly’ better than existing agents in regard to ‘sustained clinical response’; or (3) be priced at a reasonable cost when it comes to market or perhaps all three. Their utility can only be proven by clinical trials.

1. Introduction initial agent of choice to a secondary role often when there are Clostridioides (Clostridium) difficile Infection (CDI) is a major cause extremecostconsiderationsbecauseitwasshowntobeinferiorto of healthcare-associated diarrheal infection, an immediate and vancomycin for even mild and moderate disease with an increased urgent global healthcare threat and a reportable disease to recurrence rate. Fidaxomicin waselevatedasafirstlineoption[10]. National Health Safety Network (NHSN) [1] and is an element in Their focus was not based just on equivalence, but highlighted the the value-based purchasing initiative. The Centers for Medicare need for new drugs to prove superiority in sustained clinical and Medicaid Services (CMS) introduced Hospital Inpatient response (sustained cure/lower relapse rate), which is a major Value-Based Purchasing program, which are quality perfor- concernasevenwithfidaxomicintherecurrencerateat25days mance–based adjustments of up to 1% to Medicare reimburse- post-therapy is approximately 15% [11,12]. Consequently, there is ments for acute care hospitals [2]. As part of this value-based need for better therapeutic alternatives with improved efficacy, purchasing initiative, CMS reduces payment for patients diag- lower recurrence rates, and lower costs. Unfortunately, there have nosed with selected hospital-acquired infections such as been some recent expensive failures such as cadazolid and suro- C. difficile [3,4]. Over the past 15 years, there has been a 237% tomycin that did not reach their endpoints after phase 3 drug increase in CDI-related hospitalizations [5]. Primary (15–40%) and development. This highlights the question of how will a new secondary (15–30%) CDI recurrence is not infrequent including agent differentiate itself, what questions/endpoints need to be a 31% risk in patients >65 years old [6], 33% risk in immunocom- asked and how diagnostic criteria for CDI to establish study inclu- promised hosts and a twofold increased risk in patients with sion should be documented [13]. A number of new agents with renal disease [7]. These recurrences lead to an increased re- narrower spectra against normal microbiota, different targets admission in 28.3% of patients (75.2% vs. 46.9% CDI infected including effecting toxin secretion and inhibiting spore formation, vs. non-infected, respectively) [8]. The increased 30-day mortality new treatment paradigms and rationales are attempting to answer has risen to 36.3% vs. 25.7% (p < 0.004) [9]. CDI has added these questions and potentially improve patient outcomes (Tables approximately $4.5 billion in extra medical care costs and 1 and 2). patients have a 55% increased length of stay. In 2017, CDI became a quality indicator and hospitals can be penalized for 2. Methodology both readmissions and as a quality measure. The 2018 Infectious Diseases Society of America (IDSA) We searched MEDLINE, PubMed, Embase, Web of Science, Guidelines [7] have devalued the use of metronidazole from an Cochrane Central Register of Controlled Trials, and ClinicalTrials.

CONTACT Ellie J. C. Goldstein [email protected] 2021 Santa Monica Blvd #740E, Santa Monica, CA 90404, USA © 2019 Informa UK Limited, trading as Taylor & Francis Group 2 M.-C.N.TRANETAL.

3.2. DS2969b Article highlights DS-2969b (4-chloro-5-ethyl-N(3S,4R)-1-[5(2-hydroxypropan-2-yl)- ● Clostridioides (Clostridium) difficile infection (CDI) represents the most 1,3,4-thiadiazol-2-yl]-3-methyloxypiperidin-4-yl)-1H-imidazole common cause of nosocomial diarrhea worldwide. However, cur- rently approved agents for CDI therapy (metronidazole, vancomycin, -2-carboxamide 2/3 hydrate, is a novel GyrB inhibitor [27]. It and fidaxomicin) still carry a high rate of recurrence (20–40%). inhibits differently than fluoroquinolones (which binds at the ● New agents with novel mechanisms of action, narrow spectrums of enzyme–DNA interface in the cleavage–ligation active site) by activity, different targets or approaches must show superiority in relapse rates to achieve market penetration. ACX-362E, a small mole- binding to the ATP-binding site of DNA gyrase [28]. It showed cule using a new target DNA polymerase IIIC inhibition, is about to a half maximal inhibitory concentration (IC50)of20nl/mlagainst start Phase 1 trials. Early studies have suggested inhibition of DNA C. difficile. Several studies have evaluated its in vitro activity replication by ACX-362E in a mechanism that is unique to anti- C. difficile drugs. Ribaxamase (SYN-004), an orally ingested coated against C difficile.Mathuretal.foundittohaveaMIC90 of 0.06 enteric enzyme formulation, prevents microbiome disruption by µg/ml against 55 isolates, as well as a low propensity for in vitro degrading selected beta-lactam agents. resistance development [28]. Tyrrell et al. studied its activity ● RBX2260 is a commercially prepared, standardized fecal microbiota suspension packaged as a single administered enema bag. against 102 recent North American ribotyped C difficile isolates. ● Cadazolid and surotomycin were costly mistakes, failing to achieve It had an MIC50 of 0.06 μg/ml and an MIC90 of 0.125 μg/ml with final approval due to phase 3 clinical trials not meeting the primary arangeof0.03–0.125 μg/ml [29]. Using a golden Syrian hamster endpoints. Future clinical trials conducted in the pipeline agents must have consistent inclusion and testing definitions used in model with a NAP 1/027 strain, Mathur et al. suggested the high Phase 2 and Phase 3 trials as well as set considered primary and activity of DS-2969b against C. difficile,that10μg/g fecal levels secondary endpoints. would be sufficient for clearing C. difficile from the intestine [28]. This box summarizes key points contained in the article. Mathur et al. further speculated that DS2969 demonstrated promise as an oral and intravenous option for treatment of CDI. A Phase 1 placebo-controlled study of 24 healthy patient volunteers to assess the safety, tolerability, pharmacokinetics, gov for C. difficile and for agents in early stages of clinical and effects on the normal gastrointestinal microbiota of multi- development. ple daily oral ascending doses of DS-2969b in healthy subjects was recently reported [30,31]. Three sequential ascending-dose cohorts of six subjects (60 mg, 200 mg, and 400 mg for 14 days) were evaluated and 3. Investigational agents were safe and well tolerated at all doses. Nine subjects (50%) 3.1. ACX-362E reported mild events, predominantly related to the gastrointest- inal tract. Day 1 and day 14 mean plasma concentration-time ACX-362E is a novel small molecule that is using a new target, profiles found that ‘DS2969a (free form of DS-2969b) plasma DNA polymerase IIIC inhibition. DNA polymerase IIIC (pol IIIC) concentrations increased with increasing doses; however, both has been shown to be essential for replicative DNA synthesis the maximum concentration of drug in serum (Cmax) and the in aerobic, low guanine-cytosine (G-C) Gram-positive bacteria, area under the concentration-time curve (AUC) increased less than i.e. those with a low G-C ratio. Pol IIIC-specific genes of several the dose proportionally.’ While the drug is predominantly excreted such Gram-positive bacteria have been cloned and expressed in the urine (32%-54%), reasonable fecal levels were achieved [19–21], and these enzymes share a unique capacity to be on day 1 of 0.58%-1.13% and by day 14 rose to 3.21–7.89%. The inhibited by 6-anilinouracils, 2-phenylguanines (PG), and evaluation of its effect on the microbiome showed reductions in related compounds which are analogs of 2ʹ-deoxyguanosine the Clostridium coccoides and Bifidobacterium groups but little -5ʹ-triphosphate (dGTP) [22,23]. ACX-362E (formerly known as effect on Bacteroides fragilis, Clostridium leptum,andPrevotella GLS362E), a closely related dichloro-benzyl guanine inhibitor spp. suggesting a mild effect on intestinal microbiota and the of pol IIIC, emerged from an extensive discovery and synthetic protective species that maintain colonization resistance. Having effort and shows promise as a novel treatment for C. difficile just published the phase 1 data, it is unclear if the signals were diarrhea [24]. strong enough to pursue phase 2 trials. ACX-362E has been demonstrated to inhibit purified C. difficile pol IIIC with a Ki of 0.325 µM [22]. In addition, a whole cell study involving the measurement of chromoso- 3.3. LFF571 mal DNA replication demonstrated gene dosage results that suggest inhibition of DNA replication by ACX-362E in LFF571 is a semisynthetic thiopeptide that binds with high a mechanism that is unique to anti-C. difficile drugs [25]. affinity to and inhibits exogenous protein synthesis elongation ACX-362E has shown in vitro potency against 363 strains of factor EF-Tu and interferes with the ability for EF-Tu to deliver

C. difficile, with an MIC50 = 2 µg/mL [25], and in vivo activity in aminoacylated tRNA to the ribosome [32]. It possesses potent the hamster model [24]. ACX-362E has been granted Qualified in vitro activity against C.difficile (MIC range, 0.125–0.5 μg/ml;

Infectious Disease Pathogen (QIDP) status by the US FDA and MIC90 <0.25μg/ml) which was one-dilution lower than fidaxo- has received an Investigational New Drug (IND) with Phase 1 micin in the same study. LFF571 was also active against most trials beginning in December 2018 [26]. other Gram-positive anaerobes (MIC90 <0.25μg/ml), with the Table 1. Main characteristics (target, spectrum of activity, and MICs) of C. difficile treatments in development. Treatment in development Target Spectrum of activity MIC ranges against C. difficile

ACX-362E [19–21,25] Blocks the DNA replication process through inhibition of polymerase III Narrow-spectrum MIC50: 2 mg/L MIC90: 4 mg/L DS-2969B [27–29] Binds to the ATP binding site of GyrB and inhibits DNA gyrase activity Narrow-spectrum antimicrobial activity against Gram-positive bacteria, including strict MIC range of 0.015–0.06 mg/L anaerobes and facultative anaerobes LFF571 [32,33] Thiopeptide antibiotic Activity against other Gram-positive anaerobes and Gram-positive aerobes, including MIC range of 0.06–0.5 mg/L Bacterial protein synthesis disruption by inhibition of EF-Tu lactobacilli and enterococci Ridinilazole Heterocyclic antibiotic Narrow-spectrum of activity against Gram-positive pathogens including C. difficile MIC90 values of 0.125–0.25 [55,56,61,62] Bacterial DNA synthesis inhibition mg/L Ribaxamase [43,44] β-Lactamase enzyme that degrades selected β-lactam antibiotics by cleaving N/A N/A the β-lactam ring RBX2660 [67] Intestinal microbiota Broad-spectrum microbiota N/A CRS3123 [83,84] Diaryldiamine Activity against Gram-positive bacteria including Staphylococcus and Enterococcus, MIC range of 0.5–1 mg/L and Inhibition of bacterial protein synthesis (bacterial methionyl-tRNA synthetase) sparing Lactobacillus and Bifidobacterium MIC90: 1 mg/L MCB3681/MCB3837 50S ribosome subunit and DNA gyrase/topoisomerase Activity against clostridia, bifidobacteria, lactobacilli, enterococci and Staphylococcus MIC range of 0.008–0.5 mg/L [87,88] aureus Ramizol [70,71] Styrylbenzene antibiotic Narrow-spectrum MIC range ≤ 0.12–8 mg/L Mechanosensitive ion channel of large conductance (MscL) and antioxidant Ramoplanin [74– Glycolipodepsipeptide antibiotic Activity against aerobic and anaerobic Gram-positive bacteria MIC range of 0.25–0.50 mg/L

76,78,79,81] Indirect inhibition of peptidoglycan biosynthesis DRUGS INVESTIGATIONAL ON OPINION EXPERT Cadazolid [89–91] Oxazolidinone antimicrobial, containing a quinonolone pharmacophore Narrow-spectrum MIC range of 0.06–0.25 mg/L incorporated in an oxazolidinone ring Bacterial DNA and protein synthesis inhibition Surotomycin [95,96] 13-Amino acid semisynthetic lipopeptide Bactericidal activity against Gram-positive bacteria. Activity gut microbiota, including MIC90 0.125–0.25 mg/L Calcium-dependent cell membrane depolarizing agent Bifidobacterium and Lactobacillus spp. DNA = deoxyribonucleic acid; EF-Tu = elongation factor-thermo unstable; tRNA = transfer ribonucleic acid 3 4 M.-C.N.TRANETAL. Table 2. Clinical Trial information (comparator agents, trial phase, and notable findings) on C. difficile treatments in development. Treatment in Comparator Phase of clinical trials & development treatment clinical trial # Notable findings from studies ACX-362E N/A Pre-clinical ● ACX-362E has shown in vitropotency against 363 strains of C. difficile [20] ● ACX-362E Phase 1 clinical trial projected to completeethe second quarter of 2019 DS-2969B Placebo I ● DS-2969b was safe and well tolerated at all dose levels examined ● In all cohorts, sufficient fecal levels of DS-2969a were achieved within 24 h following the administration of the first dose and maintained for at least17 days [25,26] LFF571 Vancomycin II (NCT01232595) ● LFF571 was found to be more efficacious in the hamster model of CDI than vancomycin; it had a lower effective dose and fewer recurrences [14] ● Phase II trial results revealedhigher clinical response rates atthe end of treatmentwith LFF571 (90.6% vs. 78.3%), but there were alsohigher recurrence rates with LFF571 (37% vs. 31%). LFF571 treatment was non-inferior to vancomycin treatment. ● The incidence of adverse events related to LFF571 was higher than that related to vancomycin (76.1% vs. 69.2%). However, this adverse event rate needs further evaluation since the study was limited by a small enrollment [36]. Ridinilazole Fidaxomicin II (NCT02784002) ● Phase II trial demonstrated ridinilazole superiority in achieving response rates at the end of treatment (77.8% and 69.7% for ridinilazole and (Phase II only) III (NCT03595566) vancomycin, respectively), in reducing rates of recurrent CDI (14.3% and 34.8% for ridinilazole and vancomycin, respectively) and in obtaining Vancomycin sustained clinical responses (66.7% and 42.4% for ridinilazole and vancomycin, respectively) [60] ● Phase III estimated study start date 3/2019 Ribaxamase N/A II (NCT02419001 & ● 2 Phase 2a clinical studies demonstrated that ribaxamase fully degraded ceftriaxone to below the level of quantitation in the intestines of all subjects NCT02473640) in both studies [48] RBX2660 Placebo II (NCT01925417) ● A Phase 2b trial of adults with ≥2 CDI recurrences revealed that 1, but not 2, doses of RBX2660 was superior to placebo. The overall efficacy (including II (NCT02299570) open-label response) for RBX2660-treated participants was 88.8% [68] CRS3123 N/A I (NCT02106338 & ● A hamster model showed CRS3123 has long-lasting efficacy with no recurrence [85] NCT01551004) MCB3681/ N/A I ● A Phase I study showed that MCB3681 is antibacterially efficacious in vivo without affecting the Gram-negative microflora [15] MCB3837 Ramizol Vancomycin Pre-clinical ● Ramizol-treated hamsters had a 57% survival rate vs. 86% in vancomycin-treated hamsters [66] Ramoplanin Vancomycin III (FDA-approved) ● In vitro and in vivo, C. difficile spores were less often recovered from the ramoplanin-treated hamsters compared to those treated with vancomycin [79] ● A Phase II trial showed similar clinical cure rates for ramoplanin-treated vs. vancomycin-treated patients at the end of treatment (83–85% vs. 86%, respectively), but also higher rates of adverse events (nausea, vomiting, and diarrhea) [16] Cadazolid Vancomycin III (NCT01987895) ● in vitro and in vivo, cadazolid was found to be more bactericidal than vancomycin [17]and inhibit C. difficile sporulation even at sub-growth inhibitory concentrations [90] ● Phase II study revealed that patients with CDI treated with cadazolid had a lower recurrence rate compared to those receiving vancomycin (18.2–25.0% vs. 50%) and had higher sustained clinical response rates (46.7–60.0% vs. 33.3%) [93] ● A following study found that cadazolid was more effective against the C. difficile strains isolated from patients in the Phase 2 study due to MICs for cadazolid being lower than those of vancomycin, linezolid, and moxifloxacin [18] ● Phase III IMPACT 1 study met its primary endpoint, while the second Phase III study, IMPACT 2, did not. Surotomycin III (NCT01597505) ● Phase II results revealed that surotomycin-treated patients had similar clinical cure rates (92.4–86.6%) to vancomycin-treated patients (89.4%), but recurrence rates in surotomycin arms (27.9–17.2%) were lower than the vancomycin treatment group (35.6%) [97]. The sustained clinical response rates in the surotomycin groups (66.7–70.1%) at the end of study were higher than the vancomycin group (56.1%) ● Phase III results showed both the clinical cure rate and sustained clinical response rate of surotomycin-treated patients were lower than those who received vancomycin (clinical cure rate: surotomycin 79.0% vs. vancomycin 83.6%; sustained clinical response rate: surotomycin 60.6% vs. vancomycin 61.4%) – did not meet primary endpoint [97] ● Surotomycin failed to demonstrate a significant benefit over vancomycin in a parallel Phase III trial [96] EXPERT OPINION ON INVESTIGATIONAL DRUGS 5

exception of bifidobacteria and lactobacilli. It was much less the PenP protein of ~29 kDa) [38–42]. Ribaxamase is a pH- active against Gram-negative anaerobes with MICs for dependent formulation that is released in the proximal Bacteroides fragilis of 4 and 8 μg/ml, and the other species in small intestine [43,44] that has no effect in the dog model the B. fragilis group, including Bacteroides thetaiotaomicron, on the plasma pharmacokinetics of ceftriaxone [45]. In two Bacteroides ovatus,andParabacteroides (Bacteroides) distasonis, placebo-controlled Phase 1 studies, it was well tolerated as Prevotella bivia, Prevotella melaninogenica/denticola,and single doses (up to 750 mg) and multiple doses (up to Veillonella spp were even less susceptible, with an overall 300mgevery6hfor7days)[46]. There was negligible

MIC90 of 32 μg/ml. This suggests that LFF571 might have a less systemic absorption [46]. A pig model showed the addition impact on the normal gut microbiota and can help maintain of ribaxamase to ceftriaxone therapy resulted in protection colonization resistance [33]. Leeds et al. showed that sponta- of the pre-treatment microflora [47]. neous mutants with reduced susceptibility to LFF571 were Two open-label, randomized Phase 2a studies of IV ceftriax- selected in vitro in a single step, but not via serial passage [34]. one alone or with oral ribaxamase and limited use of proton After a dose ranging study comparing single and multiple pump inhibitors (PPI) enrolled subjects with functioning ileos- ascending oral doses (25 mg, 100 mg, or 200 mg every 6 h for tomies to allow easy serial sampling of their intestinal chyme. 10 days) [35] a phase 2, multicenter, randomized control trial Ceftriaxone and ribaxamase in chyme and plasma were serially compared LFF571 (200 mg four times daily) to vancomycin analyzed to examine its capacity to degrade ceftriaxone excreted (125 mg four times daily) (NCT01232595) and enrolled 72 into the intestine (registered at ClinicalTrials.gov under patients. Mullane et al. reported a LFF571 clinical cure rate of NCT02419001 and NCT02473640) [48]. Ribaxamase was well 90.6% compared to 78.3% for vancomycin with 30-day cure tolerated and fully degraded ceftriaxone to below the level of rates of 58.7% and 60.0%, respectively. However, when recur- detection in the chyme. Ribaxamase showed no effect on cef- rence rates were analyzed using toxin-confirmed cases it was triaxone plasma pharmacokinetics. The effect of the PPI esome- 19% vs 25%, respectively [36]. A number of confounding prazole co-administration did not affect ribaxamase’s ability to factors occurred within the treatment groups but were not degrade ceftriaxone excreted into the intestine. found to be significant. LFF571-treated patients tended to A proof of concept Phase 2b study of 412 patients have more potential risk factors for a poor outcome than did admitted to the hospital for lower respiratory tract infec- the vancomycin-treated patients. Older age, more first tions, compared ceftriaxone with and without ribaxamase relapses, more severe infections, less prior effective therapy, and resulted in a significantly (p = 0.045) reduced incidence and more concomitant antibiotic use were found among the of CDI by 71% as compared to placebo and tended to LFF571-treated patients. The vancomycin-treated patients decrease the incidence of antibiotic-associated diarrhea were more likely to have infections caused by the NAP1/BI/ [49]. Microbiologically, it reduced new colonization with 027 strain of C. difficile and higher use of proton pump inhi- both C. difficile and vancomycin-resistant enterococci (VRE) bitors. During the same trial, Bhansali et al. calculated phar- and reduced enterococcal mono-domination (defined as macokinetic parameters from drug concentrations measured >30% of gut microbiome) in these patients [49,50] in serum and fecal samples showing limited systemic exposure A Phase 3 study has been proposed to be initiated in with the highest observed LFF571 serum concentration of 41.7 the second half of 2019 to evaluate the potential efficacy ng/ml, but fecal levels at the end of treatment of between 107 and safety of ribaxamase in a broader patient population by and 12,900 μg/g [37]. Adverse events were similar between the including additional IV beta-lactam antibiotics with cef- the experimental arm with LFF571 and the control arm with triaxone and by enrolling patients with a variety of underlying vancomycin (76.1 vs. 69.2%, respectively). To date, no phase 3 infections [51]. The company expects the primary efficacy trial has been reported and one may speculate that although endpoint of the proposed Phase 3 clinical trial will be the the phase 2 trial was small, the possibility of the potential reduction of the incidence CDI in the ribaxamase treatment development of in vitro C. difficile LFF571 resistance may have group relative to placebo, and the primary safety endpoint will been a concern or lacking a superiority signal for relapses may be to assess mortality risk. have made this agentless attractive. Additionally, longitudinal stool samples collected from ~100 patients underwent metagenomic sequencing and the sequences were interrogated against the CARD (https://card. 3.4. Ribaxamase mcmaster.ca/) database. Over 1300 AMR genes were identified Ribaxamase (SYN-004) is a drug with a novel approach to in the gut resistome. LefSe analysis identified a significant the prevention of CDI using an orally ingested enteric- increase in placebo-treated patients compared to study coated enzyme formulation to prevent microbiome disrup- patients. qPCR analysis supported that placebo group patients tion by degrading selected β-lactam antibiotics including had more new acquisition of CfxA resistance genes and also penicillins and cephalosporins, by cleaving the β-lactam acquired new genes that expanded the antimicrobial resis- ring. The hope is to enable a patient to leave the hospital tance pool [50]. Ribaxamase reduced ceftriaxone-mediated with the same microbiome with which he/she came. It is changes in the gut resistome and has the potential to reduce concurrently co-administered with intravenous ceftriaxone, the emergence of antimicrobial resistance. a commonly used cephalosporin. Ribaxamase is a single This product has only been tested for its protective effect in amino acid substitution modification (Asp 247 [Ambler patients receiving intravenous ceftriaxone but is intended for 276] to Asn) of a class A β-lactamase (P1A, a naturally use in conjunction with intravenous penicillins and cephalos- occurring penicillinase isolated from Bacillus licheniformis, porins but not carbapenems. 6 M.-C.N.TRANETAL.

3.5. Synthetic biologics has two other products in 0.125–8 μg/mL; MIC90, 8 μg/mL) or vancomycin (MIC range, development 0.5–4 μg/ml; MIC90, 2 μg/mL) [61,62]. The highest ridinilazole MIC reported in multiple in vitro studies was 0.5 μg/ml [55]. It SYN 007 is a reformulated version of ribaxamase targeting oral β- has been shown to be less active in vitro than vancomycin and lactam antibiotics intended for use in patients treated with oral metronidazole against a wide variety of intestinal gram- beta-lactams and co-administered with oral penicillins (amoxicillin positive and gram-negative aerobes and anaerobes suggest- and amoxicillin clavulanate) [52,53].Theenzymeisareformulated ing it is sparing of the normal intestinal flora [63]. This has distal-release formulation to releaseatthesitedistaltooralanti- been confirmed in an animal model where ridinilazole admin- biotic absorption. It has been evaluated in vitro and in pig and dog istration showed ‘negligible changes’ in counts of a variety of animal models and did not interfere with oral amoxicillin absorp- bacterial groups, including Bacteroides, bifidobacteria, and lac- tion. Full data on microbiome protection analyses based on whole tobacilli [64,65]. genome shotgun sequencing of fecal DNA collected prior to and It has exhibited a prolonged post-antibiotic effect (4–20 h) after antibiotic administration is in progress. against C. difficile ribotypes 012, 027 and 078 at 10× MIC concentrations with no growth recovery following 1 h of treat- 3.6. SYN 006 ment 20× MIC [61] and significantly reduced toxin A and B concentrations even at 0.5 x MIC [56]. SYN 006 is a metallo-beta-lactamase derived from B. cereus Using a hamster model of CDI ridinilazole displayed efficacy and was produced in E. coli was identified to expand the against C. difficile ribotype 027 strains [58]. There was a 90–100% prophylactic approach to all classes of beta-lactams including survival of infected hamsters at Day 28 with a twice-daily dose of carbapenems [54]. It is intended for co-administration with ridinilazole and remained culture negative for C. difficile spores intravenous carbapenems such as ertapenem. Using an animal for a longer period than fidaxomicin-treated hamsters. model, antibiotic degradation was assessed using a bacterial Vickers et al. recently reported the results of a Phase II trial growth assay. SYN-006 (1 mg/kg, PO) displayed a broad anti- in which ridinilazole was shown non-inferior to vancomycin biotic degradation profile that included carbapenems, cepha- (15% non-inferiority margin; p = 0.0004), was well tolerated losporins, and penicillins, and was resistant to beta-lactamase and that sustained clinical response rates were 24/36 (66.7%) inhibitors. In chyme at levels of 0.5 U/g or higher, it completely and 14/33 (42.4%) for patients on ridinilazole and vancomycin, degraded meropenem in the GI tract of fistulated dogs that respectively [60]. Its adverse event profile was comparable to received meropenem (30 mg/kg, IV) and did not affect sys- vancomycin. Amongst all treated patients, 82% (41/50) and temic antibiotic levels. Chyme levels were extremely variable 80% (40/50) had an adverse event of which 16% (8/50) and due to sensitivity to acid as a non-enteric-coated liquid for- 18% (9/50) were severe in the ridinilazole and vancomycin mulation was used in this study. In a carbapenem-mediated groups, respectively. There was no study drug-related adverse microbiome disruption pig model, ertapenem significantly events that led to ridinilazole discontinuation. Based on these changed the composition of the gut microbiome and results, ridinilazole is embarking on Phase 3 studies using mediated emergence and propagation of a wide range of 200 mg orally twice a day compared to standard dose antibiotic resistance genes, including extended spectrum vancomycin. beta-lactamase genes and the carbapenemase gene, IMP-27 [47]. SYN-006 protected the pig gut microbiome from disrup- tion and attenuated antibiotic resistance [54]. 3.8. RBX2660 RBX2660 is a commercially prepared, standardized, fecal 3.7. Ridinilazole microbiota suspension preparation that is packaged into sin- Ridinilazole [2,2-bis(4-pyridyl)3H,3ʹH 5,5-bibenzimidazole], for- gle administration, ready to use enema bag [66]. In the PUNCH merly known as SMT19969, is a new non-absorbable antibac- CD study, each dose of RBX2660 was made up of 50 g of terial that acts differently and not through the classical human stool/150 mL 0.9% saline/polyethylene glycol 3350 pathways as cell wall inhibition, nor through protein, lipid, vehicle and contained ≥107 live organisms/mL of suspension RNA or DNA synthesis. In fluorescent-labeling studies using that was stored frozen at ≤−80°C. It was then thawed prior to sub-therapeutic concentrations of drug to treat C. difficile shipment to the clinical site and stored at room temperature resulted in 'a filamentous phenotype with replicated nucleoids for up to two days prior to administration to patients. along the length of the cell and no observed septum forma- The PUNCH CD study (NCT01925417) was a prospective, tion suggesting that ridinilazole may impair cell division multicenter open-label study of RBX2660 administered via [55,56]. Ridinilazole elongates C. difficile cell and inhibits spor- enema [66]. Patients eligible for study were required to have ulation, which is a unique mechanism compared to current at least two recurrent CDI episodes or at least two severe antimicrobials for treatment of C. difficile [56]. It has activity episodes resulting in hospitalization. Thirty-one of 40 enrolled against Gram-positive pathogens including C. difficile and only patients completed 6-month follow-up with an overall efficacy has minimal activity in the gut microbiota [57–59]. of 87.1% (16 with 1 dose and 11 with 2 doses). Reported Vickers et al. recently reviewed its developmental course adverse effects included GI side effects as diarrhea, flatulence, [60]. Several in vitro studies have noted ridinilazole to have abdominal pain/cramping, and constipation. Stool samples lower MICs, including ribotype 027 (MIC range, 0.06–0.25 μg/ from many of the PUNCH ID recurrent CDI study patients mL; MIC90, 0.125 μg/mL) than metronidazole (MIC range, were sequentially cultured for VRE, and this secondary analysis EXPERT OPINION ON INVESTIGATIONAL DRUGS 7

suggested that RBX2660 administration successfully converted against gram-positive aerobic and anaerobic organisms such a high percentage of VRE-positive patients to negative [67]. as C. difficile, Streptococcus spp., Staphylococcus spp., A randomized, double-blind, placebo-controlled Phase Enterococcus spp., Eubacterium spp., Lactobacillus spp., 2B trial (NTC02299570) [68] enrolled adults with 2 or more Peptostreptococcus spp., Propionibacterium spp., and CDI recurrences to receive either ‘2 doses of RBX2660 Prevotella spp. [78,79]. Ramoplanin is a glycolipodepsipeptide (group A); 2 doses of placebo (group B); or 1 dose of antibiotic with MICs ranging from 0.25 to 0.50 μg/ml [77,80]. RBX2660 followed by 1 dose of placebo (group C).’ C. difficile isolates with reduced susceptibility to vancomycin (8 Patients were followed up to 8 weeks. If a recurrence of 105 isolates) were susceptible to ramoplanin [81]. An occurred within 8 weeks, patients were eligible to receive in vitro gut and a hamster model showed that ‘ramoplanin up to 2 open-label doses of RBX2660. The primary endpoint and vancomycin were similarly effective at reducing cytotoxin was efficacy for group A compared to group B. Secondary production in the gut CDI model and in resolving symptoms in endpoints included the efficacy of group C compared to the hamster model’ [78]. group B, combined efficacy in the blinded and open-label In addition, studying the potential activity of ramoplanin phases, and safety for 24 months. The efficacy ranged from against C. difficile spores, it was noted that ramoplanin 61%, 45%, and 67%, for A, B, and C groups, respectively. adheres to the exosporium for a prolonged period so that it While the study did not meet its primary endpoint, it is available to attack newly germinating cells which would showed that a single dose of RBX2660 was superior to augment its bactericidal activity against vegetative C. difficile placebo with an overall efficacy (including open-label forms [75]. response) of 88.8%. These data are to be used for a larger A Phase 2, randomized, parallel group, multicenter, open- Phase3trial. label trial of 86 patients compared ramoplanin at 10 day RBX 7455 is another new product under early development course of 200 mg orally twice daily (n = 28) or 400 mg orally and is lyophilized, non-frozen oral capsule formulation of twice daily (n = 29) or vancomycin 125 mg orally four times recurrent CDI spore-forming organisms being developed for daily (n = 29) in CDAD [82]. The primary endpoint was a test-of the prevention of CDI [69]. It is in Phase 1 development -cure visit 1–2 weeks after the completion of the antibiotic. (NCT02981316), with a prospective, single center, two-arm The response rate for the test of cure visit 1–2 weeks after study design. It is a proof of concept dosing study of completion of antibiotic was 83% with ramoplanin 200 mg, RBX7455 for the prevention of recurrent CDI. 85% with ramoplanin at 400 mg and 86% with vancomycin. The rate of relapse was 26.3% in the ramoplanin 200 mg group, 21.7% in the ramoplanin 400 mg group and 20.8% in 3.9. Ramizol the vancomycin-treated group. It was well tolerated with side Ramizol targets the mechanosensitive ion channel of large effects similar to placebo. This trial demonstrated that ramo- conductance (MscL) in bacteria [70]. MscL does not exist in planin is efficacious with limited toxicity, but was not powered humans and protects bacterial cell wall from lysis by decreas- sufficiently to demonstrate non-inferiority to vancomycin, but ing osmotic environment. MscL opens up releases solute and larger studies are warranted. small proteins, which decreases the tension across the cell membrane, which serves as a potential target for drugs. 3.11. CRS3123 (formerly REP3123) Ramizol lowers the threshold for the MscL to open and main- tains it open longer, which slows the growth of bacteria. CRS3123, formerly known as REP3123 inhibits bacterial Ramizol demonstrates activity against clostridium difficile methionyl-tRNA synthetase (MetRS) of Gram-positive bacteria, in vitro with an MIC range of ≤0.12–8 μg/ml [71]. In including C. difficile B1/NAP1/027 strains [83]. It was active a C. difficile colitis model using Golden Syrian hamsters and against clinical strains of C. difficile with an MIC 90 of 1 μg/l strain ATCC BAA-1805, a ribotype 027 NAP-1 epidemic strain, and a range of 0.5–1 μg/ml [83,84]. an increase in survival rate of 57% for orally administered However, CRS3123 does not have any effect of major intest- ramizol at 100 mg/kg twice a day for 5 days and 86% for orally inal Gram-positive colonizers and gram-negative bacteria [84]. administered vancomycin 20 mg/kg twice a day for 5 days In a hamster gastrointestinal infection model, CRS3123 caused during the 28-day observation [72]. At 100 mg/kg four times >10-fold reduction of the sporulation of C. difficile [85]. In a day, the survival rate increased to 71%. None of the hamsters a Phase I, double-blind, placebo-controlled, dose escalation receiving ramizol had any diarrhea, which suggest little dis- study evaluated the safety and systemic exposure of ruptions to the gut flora. Ramizol has been developed as an CRS3123 after a single oral dose in 40 healthy adults it was intramuscular and subcutaneous injection to increase the half- well tolerated [86]. Its bioavailability declined with increasing life and systemic absorption [73]. Phase I studies are required dose because absorption is not proportional to the dose. to assess the safety of ramizol in healthy volunteers. Common adverse effects were decreased hemoglobin, head- ache, and abnormal urinalysis; 3.10. Ramoplanin 3.12. MCB3681/MCB3687 Ramoplanin disrupts bacterial cell wall synthesis through pep- tidoglycan binding [74–76]. MCB3837, a novel small molecule, is an injectable oxazolidinone– Oral ramoplanin is not systemically absorbed but has high quinolone combination prodrug that converts to active sub- concentrations in the feces [77]. Ramoplanin has activity stance MCB3681 [87]. In vitro, MCB3681 demonstrated an MIC 8 M.-C.N.TRANETAL. of 0.008 μg/ml to 0.5 μg/ml against 117 moxifloxacin-resistant versus vancomycin was statistically significant (P = 0.035). Yet C. difficile strains including ribotype 027 [88]. Fidaxomicin was one of the two Phase III trials failed for the initial clinical cure more active than MCB3681 with a geometric mean MIC of 0.05 primary endpoint based on a noninferiority hypothesis with μg/ml compared to MCB3681 (P = 0.0001) which had a 10% noninferiority margin compared with comparator treat- a geometric mean MIC of 0.12 μg/ml. In a Phase I trial of 12 ment (vancomycin 125 mg q6h x 10 days) [96,97]. The difference healthy male subjects, MCB3837 was found to have high con- and 95% confidence interval for initial cure for Study 1 was −4.6 centration in fecal matter and did not affect Gram-negative (−11.0, 1.9) with surotomycin 229/290 (79%) compared to van- aerobes and anaerobes, which contributes to maintaining comycin 234/280 (83.6%) and for study 2 was 1.4% (CI, −4.9, 7.6), a healthy gut microbiota. Since both cadazolid and this com- surotomycin 237/290 (83.4%) vs. vancomycin 239/280 (82.1%), pound have similar structural elements of an oxazolidinone and respectively. Recurrence and sustained cure were secondary end- a quinolone and considering the recent issues with the cadazolid points, assessed 30 days after end of treatment. Recurrence was Phase 3 trials, one should await further clinical trial evaluation of defined as a new episode of diarrhea with positive stool test for MCB3837. toxigenic C. difficile or discontinued before Day 40. The recur- rence rates, differences and Confidence Intervals reported in the papers are as follows: Study 1, surotomycin, 17.7% versus vanco- 4. Learning from costly errors – cadazolid & mycin, 21.2%, −3.5% (−10.0, 3.0); Study 2, surotomycin 19.5% surotomycin versus vancomycin, 22.4%, −2.9% (−9.5, 3.8). In retrospect, diag- Cadazolid (ACT-179,811) is a non-absorbable, narrow spectrum nostic methods, done by the study sites (EIA and/or PCR), may agent comprise an oxazolidinone pharmacophore with have included patients of true diagnostic uncertainty that could a substituted fluoroquinolone moiety [89,90]. The oxazolidinone have led to an underestimate of efficacy magnitude. Since sur- moiety inhibited protein synthesis and the fluoroquinolone inhib- otomycin did not demonstrate ‘non-inferiority ’ to vancomycin ited DNA synthesis. It was noted to be highly active against for CDI clinical response at end of therapy for one study and did C. difficile both in vitro and in animal models and showed biologi- not show superiority for reduction in recurrence or sustained cal effects on both C. difficile toxin production and spore forma- clinical response differences, its development was discontinued. tion [91]. A Phase 2 multicenter, randomized, dose ranging, One must consider potential variables that might have control trial of three different doses of cadazolid compared with affected outcome evaluation including endpoint definitions, vancomycin enrolled 84 patients and did not meet its endpoint patient selection as regards to testing methods used to estab- due to rigid definitions of diarrhea and cure. Resolution of diarrhea lish both diagnosis and that of relapse/failure (PCR vs EIA vs was defined as ≤2 semi-formed or formed stools and no liquid or cytotoxic assay). For instance, for surotomycin, there were dif- unformed stools for two consecutive 24-h periods and neither ferences in the trial design between the Phase 2 and 3 studies. cadazolid nor the vancomycin comparator reached the primary The inclusion criteria differed (Phase 2 trial: ≥4 unformed bowel endpoint [92,93]. However, this antibiotic was moved into Phase 3 movements (UBMs) were required for inclusion; Phase 3 trial: ≥3 because of demonstrated superiority to vancomycin for recur- UBMs) the definition of cure differed (phase 2 trial: defined as rence and sustained clinical cure. Two Phase 3 trials (IMPACT <4 UBMs per 24-h period for at least 2 consecutive days; phase 3 I and II, NCT01987895 and NCT01983683) that compared the trial: ≤2 UBMs per 24-h period for at least 2 consecutive days), efficacy of cadazolid 250 mg twice daily to vancomycin 125 mg and the follow-up period differed potentially leading to differ- four times daily also failed their endpoints. These studies devel- ent sustained clinical response rates (Phase 2 trial: 28 days; oped a new patient-reported outcome questionnaire for symp- Phase 3 trial: 30–40 days). Furthermore, in both the Phase 2 tomsofCDIaswellasanInvestigator’s assessment of clinical and Phase 3 surotomycin trials, CDI diagnosis required only one response (ICR) rate and sustained response rate (ISR Rate). toxin-positive result. The most current CDI diagnosis recom- Results have yet to be published, but after Actelion was acquired mendations include a 2-step procedure. by Johnson and Johnson, that company noted that in two trials designed to be identical, cadazolid chalked up one success and one failure, after which they discontinued current development as 5. Conclusion it was uncertain if cadazolid as effective as vancomycin [94]. The key finding of our paper is that currently approved anti- Surotomycin is a minimally absorbed lipopeptide agent with microbials for CDI have limitations, with a dire need for new limited activity against gram-negative and facultative anaerobes agents and approaches required. In comparison with the cur- of the microbiome and took a microbiome sparing approach rently available CDI treatment agents, the various novel drugs [95]. In the Surotomycin Phase II trials, the recurrence rate was: reviewed in this paper may offer several advantages. However, 17.2% vs 35.6%, for surotomycin at the higher of the two studied since these new compounds are in early development, no doses and vancomycin, respectively (P = 0.035) [96]. The primary conclusions can be reached until Phase 3 clinical trials are endpoint was resolution of diarrhea sustained through 2 days completed and analyzed. after CDI therapy ended and no need for additional antibiotics. Clinical cure rates were similar among treatment groups (92.4% for surotomycin 125 mg twice daily, 86.6% for surotomycin 6. Expert opinion 250 mg twice daily and 89.4% for vancomycin). Recurrence 6.1. Call for Novel Treatment Approaches rates were 27.9% for surotomycin 125 mg twice daily, 17.2% for surotomycin 250 mg twice daily and 35.6% for vancomycin. The We previously noted the failure of two promising agents (suroto- lower recurrence rate with surotomycin 250 mg twice daily mycin and cadazolid) due to a variety of methodological problems EXPERT OPINION ON INVESTIGATIONAL DRUGS 9 which is hopefully overcome by better trial design for these newer apparent that almost all patients respond to initial therapy agents. The ultimate goal is to have a variety of effective but that the commanding issue is recurrence as the rates are approaches in the treatment of CDI and the prevention of relapse. 20–40% for the first episode. The need for a variety of approaches is congruent with the current Recurrence is more common in healthcare-associated infec- trend for personalized medicine. It is of import that these new tions than with community-onset infections [100] and ~25% of agents offer a variety of approaches from fecal transplant, to patients were readmitted within the first 30 days [8] with enzymatic inactivation to molecules that work using different escalating risk of recurrence that increases to ~40–65% with mechanisms of action. Since the clinical problem of CDI is ubiqui- each successive episode [101]. Consequently, there is need for tous and urgent, their potential to decrease disease burden and more effective drugs and approaches. The 2018 IDSA/SHEA improve therapeutic outcome is equally great. Of note, we cov- C. difficile guidelines have suggested not only new diagnostic ered only drugs and did not review monoclonal antibodies or paradigms to better identify patients with infection rather other new potential biologics that are under development. It is than colonization but also demoted metronidazole as a first also possible that a bundle approach may be required to over- line agent and raised fidaxomicin to first-line status aside come the worldwide burden of CDI. vancomycin [7]. Chopra et al. stated that patients deemed at low risk for recurrence should receive vancomycin (or metro- nidazole when cost is an issue), while those at higher risk of 6.2. Pros and Cons recurrence would benefit from fidaxomicin treatment [102]. The favorable pharmacokinetic and pharmacodynamic profile, While the sustained clinical response to fidaxomicin when narrow-spectrum activity against C. difficile that is associated compared to vancomycin was statistically significant (15.4% with a low impact on the gut microbiota composition, and the vs 25.3% (p = 0.0002 [11] (p = 0.005 [12])), there was still a 15% inhibitory activity on C. difficile sporulation and toxins produc- recurrence rate. tion are among the most encouraging attributes of these How does one improve on this situation? Drugs should be compounds. The ability to prevent C. difficile sporulation developed that have less impact on the protective normal exhibited by several novel agents, such as ridinilazole, flora, have lower C. difficile MICs proton pump inhibitors. CRS3123, and ramoplanin suggests that these compounds During the same trial, Bhansali et al. calculated pharmacoki- may not only reduce CDI recurrence, but may also reduce netic, somehow stop toxin production by earlier inactivation C. difficile environment persistence. This would, in effect, of the exponential log phase growth and sporulation (germi- reduce the transmission of CDI in the hospital setting and nation rates and outgrowth efficiencies) or uses a new more CDI healthcare-associated acquisition. potent mechanism of action or target binding. Drug efficacy However, there are various drawbacks to these novel and effect of sub-therapeutic concentrations has been incon- agents that need to be addressed. Cadazolid and surotomycin sistent especially the drug effect on tcdA and tcdB production, failed to achieve a significant benefit over currently approved particularly during the early non-stationary stages of growth. CDI agents; LFF571 failed to reduce the CDI recurrence rate at This could possibly be related to recurrence and should be the end of treatment, and ramoplanin displaying a high a consistent parameter of evaluation. What effect new drugs impact on several phila of the gut microbiota. may have on binary toxin that potentially enhances colonic inflammation and enhancing virulence remains to be studied. Alternatively, new paradigms for therapy such as toxin target 6.3. Need for Consistency in Clinical Trial Endpoints binding, antimicrobial inactivation in the GI tract, probiotics, As noted previously, there are currently three drugs (fidaxo- improved Infection Prevention coupled with Antimicrobial micin, metronidazole, and vancomycin) currently approved for Stewardship Programs (ASPs) and perhaps instituting CDI. While the initial clinical response of these agents is gen- a ‘bundle approach’ should be sought. erally acceptable, the commanding issue is recurrence as the Of note, a European Union (EU) wide survey of 1,047 found rates are 20–40% for the first episode. The novel molecules that 97.3% of respondents thought that ‘recurrent CDI is under development for CDI present promising characteristics a strong contributor to poor clinical outcomes, increased in comparison to existing agents approved for CDI. However, length of hospital stay and increased costs’ and 90.7% of future clinical trials conducted in the pipeline agents must respondents agreed that ‘treatment decisions for both initial have consistent inclusion and testing definitions used in infection and recurrent CDI should take into account the Phase 2 and Phase 3 trials as well as set considered primary impact on resource utilization and wider societal costs.’ [103] and secondary endpoints, which could help with preventing In this new era of drug development, therapies similar to costly mistakes. Hopefully, in the near future, these new mole- current approved agents will be insufficient and one hopes cules will be effective at not only fight CDI but also preventing that these newer agents will enhance our armamentarium to this disease. alter the severity and recurrence of CDI. Future studies of groups at high risk of disease and recurrence such as the compromised host are also warranted. 6.4. Issues of initial clinical response vs. relapse From 2009 to 2013 there has been an increase in attributable mortality and length of hospital stay due to CDI [98,99] that Funding has resulted in marked increased healthcare costs. While CDI related hospitalizations increased by 237% it has become This paper is not funded. 10 M.-C. N. TRAN ET AL.

Declaration of interest 11. Louie TJ, Miller MA, Mullane KM, et al. Fidaxomicin versus vanco- mycin for Clostridium difficile infection. N Engl J Med. 2011 Feb EJC Goldstein has served on Advisory boards for Merck & Co, Bayer 3;364(5):422–431. PubMed PMID: 21288078; eng. Pharmaceuticals, BioK+, Sanofi-Adventis, Summit Corp. plc, Cutis • Shows that although fidaxomicin, one of the newest agents to Pharmaceuticals, Kindred Healthcare Corp., Novartis, Sankyo-Daichi, obtain FDA approval for CDI, was associated with Paratek Pharma, and Shionogi Inc. EJC Goldstein has also been on the a significantly lower rate of CDI recurrence, recurrence still Speakers’ bureau for Bayer Inc., Merck & Co, Medicines Co., Allergan Inc occurred in over 15% of patients. and has received research grants from Bayer Inc., Cutis Pharmaceuticals, 12. Cornely OA, Crook DW, Esposito R, et al. Fidaxomicin versus vancomy- Entasis Therpaeutics, Merck & Co., Micromyx LLC, Parateck cin for infection with Clostridium difficile in Europe, Canada, and the Pharmaceuticals, Spero Therpaeutics, Tetraphase Inc. The authors have USA: a double-blind, non-inferiority, randomised controlled trial. Lancet no other relevant affiliations or financial involvement with any organiza- Infect Dis. 2012 Apr;12(4):281–289. PubMed PMID: 22321770; eng. tion or entity with a financial interest in or financial conflict with the 13. Polage CR, Gyorke CE, Kennedy MA, et al. Overdiagnosis of Clostridium subject matter or materials discussed in the manuscript. This includes difficile Infection in the Molecular Test Era. JAMA Intern Med. 2015 employment, consultancies, honoraria, stock ownership or options, expert Nov;175(11):1792–1801. PubMed PMID: 26348734; PubMed Central testimony, grants or patents received or pending, or royalties. PMCID: PMCPMC4948649. eng. •• Reveals the importance of appropriate diagnostic testing which can confound clinical trials. Reviewer disclosures 14. Trzasko A, Leeds JA, Praestgaard J, et al. Efficacy of LFF571 in a hamster model of Clostridium difficile infection. Antimicrob Agents Chemother. Peer reviewers on this manuscript have no relevant financial or other 2012 Aug;56(8):4459–4462. PubMed PMID: 22644020; PubMed Central relationships to disclose. PMCID: PMCPMC3421564. 15. Dalhoff A, Rashid MU, Kapsner T, et al. Analysis of effects of MCB3681, the antibacterially active substance of prodrug MCB3837, on human References resident microflora as proof of principle. Clin Microbiol Infect. 2015 Aug;21(8):767 e1–4. PubMed PMID: 26047854. Papers of special note have been highlighted as either of interest (•)orof 16. Fulco P, Wenzel RP. Ramoplanin: a topical lipoglycodepsipeptide considerable interest (••) to readers. antibacterial agent. Expert Rev Anti Infect Ther. 2006 Dec;4 1. Zimlichman E, Henderson D, Tamir O, et al. Health care–associated (6):939–945. PubMed PMID: 17181409. infections: A meta-analysis of costs and financial impact on the us 17. Locher HH, Seiler P, Chen X, et al. In vitro and in vivo antibacterial health care system. JAMA Intern Med. 2013;173(22):2039–2046. evaluation of cadazolid, a new antibiotic for treatment of 2. Centers for Medicare & Medicaid Services (CMS). Medicare program; Clostridium difficile infections. Antimicrob Agents Chemother. hospital inpatient value-based purchasing program. Final rule. Fed 2014;58(2):892–900. PubMed PMID: 24277020; PubMed Central Regist. 2011 May6;76(88):26490-26547. PubMed Central PMCID: PMCID: PMCPMC3910819. PMC21548401 18. Knight-Connoni V, Mascio C, Chesnel L, et al. Discovery and devel- 3. Centers for Medicare and Medicaid Services: Medicare Learning opment of surotomycin for the treatment of Clostridium difficile. Network. Hospital Value-Based Purchasing; 2017. Available from: J Ind Microbiol Biotechnol. 2016 Mar;43(2–3):195–204.. PubMed https://www.cms.gov/Outreach-and-Education/Medicare-Learning- PMID: 26670919. Network-MLN/MLNProducts/downloads/Hospital_VBPurchasing_ 19. Hammond RA, Barnes MH, Mack SL, et al. Bacillus subtilis DNA Fact_Sheet_ICN907664.pdf [Cited 2018 Dec 1]. polymerase III: complete sequence, overexpression, and character- 4. Stone PW, Glied SA, McNair PD, et al. CMS changes in reimburse- ization of the polC gene. Gene. 1991 Feb 1;98(1):29–36. PubMed ment for HAIs: setting a research agenda. Med Care. 2010 May;48 PMID: 1901559; eng. (5):433–439. PubMed PMID: 20351584; PubMed Central PMCID: 20. Pacitti DF, Barnes MH, Li DH, et al. Characterization and overexpres- PMCPMC2881841. sion of the gene encoding Staphylococcus aureus DNA polymerase 5. Peery AF, Dellon ES, Lund J, et al. Burden of gastrointestinal disease III. Gene. 1995 Nov 7;165(1):51–56. PubMed PMID: 7489915; eng. in the United States: 2012 update. Gastroenterology. 2012 Nov;143 21. Foster KA, Barnes MH, Stephenson RO, et al. DNA polymerase III of (5):1179–1187 e3. PubMed PMID: 22885331; PubMed Central Enterococcus faecalis: expression and characterization of recombi- PMCID: PMCPMC3480553. nant enzymes encoded by the polC and dnaE genes. Protein Expr 6. Wilcox MH, Gerding DN, Poxton IR, et al. Bezlotoxumab for preven- Purif. 2003 Jan;27(1):90–97. PubMed PMID: 12509989; eng. tion of recurrent clostridium difficile infection. N Engl J Med. 2017 22. Torti A, Lossani A, Savi L, et al. Clostridium difficile DNA polymerase Jan 26;376(4):305–317. PubMed PMID: 28121498. IIIC: basis for activity of antibacterial compounds. Curr Enzyme 7. McDonald LC, Gerding DN, Johnson S, et al. Clinical Practice Inhib. 2011 Oct;7(3):147–153. PubMed PMID: 22844265; PubMed Guidelines for Clostridium difficile Infection in Adults and Children: Central PMCID: PMCPMC3404731. eng. 2017 Update by the Infectious Diseases Society of America (IDSA) and 23. Xu WC, Wright GE, Brown NC, et al. 7-Alkyl-N(2)-substituted-3-dea- Society for Healthcare Epidemiology of America (SHEA). Clin Infect zaguanines. Synthesis, DNA polymerase III inhibition and antibac- Dis. 2018 Mar 19;66(7):e1–e48. PubMed PMID: 29462280; PubMed terial activity. Bioorg Med Chem Lett. 2011 Jul 15;21 Central PMCID: PMCPMC6018983. eng. (14):4197–4202. PubMed PMID: 21684746; PubMed Central •• Updated CDI guidelines from IDSA/SHEA revealing both diag- PMCID: PMCPMC3128661. eng. nostic challenges and new treatment suggestions. 24. Dvoskin S, Xu WC, Brown NC, et al. A novel agent effective against 8. Olsen MA, Yan Y, Reske KA, et al. Impact of Clostridium difficile Clostridium difficile infection. Antimicrob Agents Chemother. 2012 recurrence on hospital readmissions. Am J Infect Control. 2015 Apr Mar;56(3):1624–1626. PubMed PMID: 22203600; PubMed Central 1;43(4):318–322. PubMed PMID: 25690876; eng. PMCID: PMCPMC3294905. 9. Olsen MA, Yan Y, Reske KA, et al. Recurrent Clostridium difficile 25. van Eijk E, Boekhoud IM, Kuijper EJ, et al. Genome location dictates infection is associated with increased mortality. Clin Microbiol the transcriptional response to sub-inhibitory concentrations of Infect. 2015 Feb;21(2):164–170. PubMed PMID: 25658560. PolC-inhibitors in Clostridium difficile. bioRxiv. 2018. DOI:10.1101/ 10. Johnson S, Louie TJ, Gerding DN, et al. Vancomycin, metronidazole, 362137. or tolevamer for Clostridium difficile infection: results from two 26. Acurx Pharmaceuticals. ACX-362E; Available from: http://www. multinational, randomized, controlled trials. Clin Infect Dis. 2014 prnewswire.com/news-releases/acurx-pharmaceuticals-to-enter- Aug 1;59(3):345–354. PubMed PMID: 24799326; eng. clinical-trials-with-first-in-class-antibiotics-to-treat-selected-gram- • Demonstrates that metronidazole is inferior to vancomycin positive-bacterial-infections-300724541.html?tc=eml_cleartime with higher relapse rates in all disease severities. [Cited 2018 Nov 18]. EXPERT OPINION ON INVESTIGATIONAL DRUGS 11

27. Kumar M, Mathur T, Joshi V, et al. Effect of DS-2969b, a novel GyrB gut microflora of healthy subjects during intravenous administra- inhibitor, on rat and monkey intestinal microbiota. Anaerobe. 2018 tion of ampicillin. Antimicrob Agents Chemother. 2009 Jun;53 Jun;51:120–123. PubMed PMID: 29758524. (6):2455–2462. PubMed PMID: 19307374; PubMed Central PMCID: 28. Mathur T, Barman TK, Kumar M, et al. In Vitro and In Vivo Activities of PMCPMC2687246. eng. DS-2969b, a Novel GyrB Inhibitor, against Clostridium difficile. 42. Stiefel U, Nerandzic MM, Koski P, et al. Orally administered Antimicrob Agents Chemother. 2018 Apr;62(4). PubMed PMID: beta-lactamase enzymes represent a novel strategy to prevent 29439962; PubMed Central PMCID: PMCPMC5913969. DOI:10.1128/ colonization by Clostridium difficile. J Antimicrob Chemother. AAC.02157-17. 2008 Nov;62(5):1105–1108. PubMed PMID: 18693236; eng. 29. Tyrrell KL, Citron DM, Merriam CV, et al. In vitro activity of DS-2969b 43. Kaleko M, Bristol JA, Hubert S, et al. Development of SYN-004, an and comparator antimicrobial agents against Clostridioides oral beta-lactamase treatment to protect the gut microbiome from (Clostridium) difficile, methicillin-resistant Staphylococcus aureus, antibiotic-mediated damage and prevent Clostridium difficile and other anaerobic bacteria. Anaerobe. 2018. DOI:10.1016/j. infection. Anaerobe. 2016 Oct;41:58–67. PubMed PMID: 27262694; anaerobe.2018.04.010. eng. 30. Dennie J, Vandell AG, Inoue S, et al. A Phase I, Single-Ascending- 44. Bristol A, Hubert S, Hofmann F, et al. Formulation development of Dose Study in Healthy Subjects to Assess the Safety, Tolerability, SYN-004 (ribaxamase) oral solid dosage form, a beta-lactamase to Pharmacokinetics, and Pharmacodynamics of DS-2969b, a Novel prevent intravenous antibiotic-associated dysbiosis of the colon. GyrB Inhibitor. J Clin Pharmacol. 2018 May 10 PubMed PMID: Int J Pharm. 2017 Dec 20;534(1–2):25–34. PubMed PMID: 28986322; 29746725. DOI:10.1002/jcph.1151. eng. 31. Vandell AG, Inoue S, Dennie J, et al. Phase 1 Study To Assess the 45. Kokai-Kun JF, Bristol JA, Setser J, et al. Nonclinical safety assess- Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of ment of SYN-004: an oral beta-lactamase for the protection of the Multiple Oral Doses of DS-2969b, a Novel GyrB Inhibitor, in Healthy gut microbiome from disruption by biliary-excreted, intravenously Subjects. Antimicrob Agents Chemother. 2018 May;62(5). PubMed administered antibiotics. Int J Toxicol. 2016 May;35(3):309–316. PMID: 29439973; PubMed Central PMCID: PMCPMC5923173. eng. PubMed PMID: 26700136; eng. DOI:10.1128/aac.02537-17. 46. Roberts T, Kokai-Kun JF, Coughlin O, et al. Tolerability and pharma- 32. Leeds JA, Sachdeva M, Mullin S, et al. Mechanism of action of and cokinetics of SYN-004, an orally administered beta-lactamase for mechanism of reduced susceptibility to the novel anti-Clostridium the prevention of Clostridium difficile-associated disease and difficile compound LFF571. Antimicrob Agents Chemother. 2012 antibiotic-associated diarrhea, in two phase 1 studies. Clin Drug Aug;56(8):4463–4465. PubMed PMID: 22644023; PubMed Central Investig. 2016 Sep;36(9):725–734. 10.1007/s40261-016-0420-0. PMCID: PMCPMC3421628. eng. PubMed PMID: 27283946; eng. 33. Citron DM, Tyrrell KL, Merriam CV, et al. Comparative in vitro 47. Connelly S, Bristol JA, Hubert S, et al. SYN-004 (ribaxamase), an oral activities of LFF571 against Clostridium difficile and 630 other beta-lactamase, mitigates antibiotic-mediated dysbiosis in intestinal strains of aerobic and anaerobic bacteria. Antimicrob a porcine gut microbiome model. J Appl Microbiol. 2017 Feb 28 Agents Chemother. 2012 May;56(5):2493–2503. PubMed PMID: PubMed PMID: 28245091. DOI:10.1111/jam.13432. 22290948; PubMed Central PMCID: PMCPMC3346664. eng. 48. Kokai-Kun JF, Roberts T, Coughlin O, et al. The Oral beta-Lactamase 34. Leeds JA. Antibacterials Developed to Target a Single Organism: SYN-004 (Ribaxamase) Degrades Ceftriaxone Excreted into the mechanisms and Frequencies of Reduced Susceptibility to the Novel Intestine in Phase 2a Clinical Studies. Antimicrob Agents Anti-Clostridium difficile Compounds Fidaxomicin and LFF571. Cold Chemother. 2017 Mar;61(3). PubMed PMID: 28052855; PubMed Spring Harb Perspect Med. 2016 Feb 1;6(2):a025445.. PubMed PMID: Central PMCID: PMCPMC5328510. eng. DOI:10.1128/aac.02197-16. 26834162; PubMed Central PMCID: PMCPMC4743069. eng. 49. Kokai-Kun J, Roberts T, Coughlin O, et al. SYN-004 (ribaxamase) 35. Ting LS, Praestgaard J, Grunenberg N, et al. A first-in-human, prevents New Onset Clostridium difficile Infection by Protecting randomized, double-blind, placebo-controlled, single- and the Integrity Gut Microbiome in a Phase 2b Study. Open Forum multiple-ascending oral dose study to assess the safety and Infect Dis. 2017FallOct04; 4(Suppl 1):S12–S12.. PubMed PMID: tolerability of LFF571 in healthy volunteers. Antimicrob Agents PMC5631845 Chemother. 2012 Nov;56(11):5946–5951. PubMed PMID: 50. Kokai-Kun NJF, Le C, Trout K, et al. SYN-004 (ribaxamase) reduces 22964250; PubMed Central PMCID: PMCPMC3486596. eng. the Emergence of Antimicrobial Resistance in the Gut Microbiome 36. Mullane K, Lee C, Bressler A, et al. Multicenter, randomized clinical of Patients Treated with Ceftriaxone. San Francisco, CA: ID Week; trial to compare the safety and efficacy of LFF571 and vancomycin 2018. for Clostridium difficile infections. Antimicrob Agents Chemother. • Coadministration of ribaxamase with IV β-lactam antibiotics 2015 Mar;59(3):1435–1440. PubMed PMID: 25534727; PubMed may protect the microbiome and may help limit the emer- Central PMCID: PMCPMC4325808. eng. gence of antibiotic resistance induced by these antibiotics. 37. Bhansali SG, Mullane K, Ting LS, et al. Pharmacokinetics of LFF571 and 51. Synthetic Biologics Announces Positive Outcome of End-of-Phase 2 vancomycin in patients with moderate Clostridium difficile infections. Meeting with FDA on SYN-004 (ribaxamase) Development: Antimicrob Agents Chemother. 2015 Mar;59(3):1441–1445. PubMed Synthetic Biologics; 2018 updated 2018;Nov 25, 2018 Nov 21, PMID: 25534724; PubMed Central PMCID: PMCPMC4325791. eng. 2018;Nov 21, 2018;Nov 25. Available from: https://www.synthetic 38. Harmoinen J, Mentula S, Heikkila M, et al. Orally administered biologics.com/news-media/press-releases/detail/266/synthetic- targeted recombinant Beta-lactamase prevents ampicillin-induced biologics-announces-positive-outcome-of selective pressure on the gut microbiota: a novel approach to 52. Kaleko M, Furlan-Freguia C, Subramanian P, et al. Orally delivered reducing antimicrobial resistance. Antimicrob Agents Chemother. beta-lactamase prevents gut microbiome dysbiosis caused by IV 2004 Jan;48(1):75–79. PubMed PMID: 14693521; PubMed Central and oral antibiotics and mitigates propagation of antibiotic resis- PMCID: PMCPMC310163. eng. tance in porcine and canine models. Poster Sa1839. Digestive 39. Harmoinen J, Vaali K, Koski P, et al. Enzymic degradation of a Disease Week 2018 2018. beta-lactam antibiotic, ampicillin, in the gut: a novel treatment 53. Connelly S, Furlan-Freguia C, Subramanian P, et al. Gut Antibiotic modality. J Antimicrob Chemother. 2003 Feb;51(2):361–365. Inactivation by Β-Lactamases Is Intended to Prevent Microbiome PubMed PMID: 12562703; eng. Damage and Attenuate Antibiotic Resistance in Large Animal 40. Pitout JD. IPSAT P1A, a class A beta-lactamase therapy for the preven- Models. Open Forum Infect Dis. 2017;4(Suppl_1):S232–S233. tion of penicillin-induced disruption to the intestinal microflora. Curr 54. Kaleko M, Furlan-Freguia C, Subramanian P, et al. SYN-006, a Novel Opin Invest Drugs. 2009 Aug;10(8):838–844. PubMed PMID: 19649928; Carbapenemase, Intended to Protect the Gut Microbiome from eng. Antibiotic-Mediated Damage May Also Reduce Propagation of 41. Tarkkanen AM, Heinonen T, Jogi R, et al. P1A recombinant Carbapenem-Resistant Pathogens. Open Forum Infect Dis. 2017;4 beta-lactamase prevents emergence of antimicrobial resistance in (Suppl_1):S231–S232. 12 M.-C. N. TRAN ET AL.

55. Vickers RJ, Tillotson G, Goldstein EJ, et al. Ridinilazole: a novel 68. Dubberke ER, Lee CH, Orenstein R, et al. Results from a Randomized therapy for Clostridium difficile infection. Int J Antimicrob Agents. Placebo-Controlled Clinical Trial of a RBX2660-a Microbiota-based 2016 Aug;48(2):137–143. PubMed PMID: 27283730. Drug for the Prevention of Recurrent Clostridium difficile Infection. • A good review of a new promising agent now in Phase 3 Clin Infect Dis. 2018 Mar 29 PubMed PMID: 29617739. DOI:10.1093/ clinical trials. cid/ciy259. 56. Basseres E, Endres BT, Khaleduzzaman M, et al. Impact on toxin 69. Inc. MCaR. Treatment of Recurrent Clostridium Difficile Infection production and cell morphology in Clostridium difficile by ridi- With RBX7455 (ClinicalTrials.gov Identifier: NCT02981316) updated nilazole (SMT19969), a novel treatment for C. difficile infection. 2018;Nov 25, 2018 Feb 22, 2018;Nov 22, 2018;Nov 25. Available J Antimicrob Chemother. 2016 May;71(5):1245–1251. PubMed from: https://clinicaltrials.gov/ct2/show/NCT02981316 PMID: 26895772; PubMed Central PMCID: PMCPMC4830417. 70. Iscla I, Wray R, Blount P, et al. A new antibiotic with potent activity 57. Goldstein EJ, Citron DM, Tyrrell KL. Comparative in vitro activities targets MscL. J Antibiot (Tokyo). 2015 Jul;68(7):453–462. PubMed of SMT19969, a new antimicrobial agent, against 162 strains PMID: 25649856; PubMed Central PMCID: PMCPMC4430313. from 35 less frequently recovered intestinal Clostridium species: 71. Wolfe C, Pagano P, Pillar CM, et al. Comparison of the in vitro implications for Clostridium difficile recurrence. PubMed PMID: antibacterial activity of Ramizol, fidaxomicin, vancomycin and 24247123; PubMed Central PMCID: PMCPMC3910813 Antimicrob metronidazole against 100 clinical isolates of Clostridium difficile Agents Chemother. 2014;582:1187–1191. by broth microdilution. Diagn Microbiol Infect Dis. 2018. 58. Sattar A, Thommes P, Payne L, et al. SMT19969 for Clostridium DOI:10.1016/j.diagmicrobio.2018.06.002. difficile infection (CDI): in vivo efficacy compared with fidaxomicin 72. Rao S, Prestidge CA, Miesel L, et al. Preclinical development of and vancomycin in the hamster model of CDI. J Antimicrob Ramizol, an antibiotic belonging to a new class, for the treatment Chemother. 2015;70(6):1757–1762. PubMed PMID: 25652749; of Clostridium difficile colitis. J Antibiot (Tokyo). 2016 Dec;69 PubMed Central PMCID: PMCPMC4498292. (12):879–884. PubMed PMID: 27189122; PubMed Central PMCID: 59. Weiss W, Pulse M, Vickers R. In vivo assessment of SMT19969 in PMCPMC5399159. a hamster model of clostridium difficile infection. Antimicrob 73. Wright L, Rao S, Thomas N, et al. Ramizol((R)) encapsulation into Agents Chemother. 2014 Oct;58(10):5714–5718.. PubMed PMID: extended release PLGA micro- and nanoparticle systems for sub- 25022586; PubMed Central PMCID: PMCPMC4187990. cutaneous and intramuscular administration: in vitro and in vivo 60. Vickers RJ, Tillotson GS, Nathan R, et al. Efficacy and safety of ridinilazole evaluation. Drug Dev Ind Pharm. 2018 Sep;44(9):1451–1457. compared with vancomycin for the treatment of Clostridium difficile PubMed PMID: 29619851. infection: a phase 2, randomised, double-blind, active-controlled, 74. Cavalleri B, Pagani H, Volpe G, et al. A-16686, a new antibiotic non-inferiority study. Lancet Infect Dis. 2017 Jul;17(7):735–744. from Actinoplanes. I. Fermentation, isolation and preliminary PubMed PMID: 28461207; PubMed Central PMCID: PMCPMC5483507. physico-chemical characteristics. J Antibiot (Tokyo). 1984 Apr;37 61. Corbett D, Wise A, Birchall S, et al. In vitro susceptibility of (4):309–317. PubMed PMID: 6547132. Clostridium difficile to SMT19969 and comparators, as well as the 75. McCafferty DG, Cudic P, Frankel BA, et al. Chemistry and biology of killing kinetics and post-antibiotic effects of SMT19969 and com- the ramoplanin family of peptide antibiotics. Biopolymers. 2002;66 parators against C. difficile. J Antimicrob Chemother. 2015;70 (4):261–284. PubMed PMID: 12491539. (6):1751–1756. PubMed PMID: 25652750; PubMed Central PMCID: 76. Boger DL. Vancomycin, teicoplanin, and ramoplanin: synthetic and PMCPMC4498293. mechanistic studies. Med Res Rev. 200109/2011;21(5):356–381.. 62. Baines SD, Crowther GS, Freeman J, et al. SMT19969 as PubMed Central PMCID: PMC11579438. a treatment for Clostridium difficile infection: an assessment of 77. Farver DK, Hedge DD, Lee SC. Ramoplanin: a lipoglycodepsipeptide antimicrobial activity using conventional susceptibility testing antibiotic. Ann Pharmacother. 2005 May;39(5):863–868.. PubMed and an in vitro gut model. J Antimicrob Chemother. 2015 PMID: 15784805. Jan;70(1):182–189. PubMed PMID: 25190720; PubMed Central 78. Citron DM, Merriam CV, Tyrrell KL, et al. In Vitro Activities of PMCID: PMCPMC4267497. Ramoplanin, Teicoplanin, Vancomycin, Linezolid, Bacitracin, and 63. Goldstein EJ, Citron DM, Tyrrell KL, et al. Comparative in vitro Four Other Antimicrobials against Intestinal Anaerobic Bacteria. activities of SMT19969, a new antimicrobial agent, against Antimicrob Agents Chemother. 2003;47(7):2334–2338. Clostridium difficile and 350 gram-positive and gram-negative 79. Freeman J, Baines SD, Jabes D, et al. Comparison of the efficacy of aerobic and anaerobic intestinal flora isolates. Antimicrob Agents ramoplanin and vancomycin in both in vitro and in vivo models of Chemother. 2013 Oct;57(10):4872–4876. PubMed PMID: 23877700; clindamycin-induced Clostridium difficile infection. J Antimicrob PubMed Central PMCID: PMCPMC3811411. Chemother. 2005 Oct;56(4):717–725. PubMed PMID: 16143709. 64. Skraban J, Dzeroski S, Zenko B, et al. Gut microbiota patterns 80. Kraus CN, Lyerly MW, Carman RJ. Ambush of Clostridium difficile associated with colonization of different Clostridium difficile spores by ramoplanin: activity in an in vitro model. Antimicrob ribotypes. PLoS One. 2013;8(2):e58005. PubMed PMID: 23469128; Agents Chemother. 2015 May;59(5):2525–2530.. PubMed PMID: PubMed Central PMCID: PMCPMC3585249. 25691641; PubMed Central PMCID: PMCPMC4394778. 65. Louie TJ, Emery J, Krulicki W, et al. OPT-80 eliminates Clostridium 81. Pelaez T, Alcala L, Alonso R, et al. In vitro activity of ramoplanin against difficile and is sparing of bacteroides species during treatment of Clostridium difficile, including strains with reduced susceptibility to C. difficile infection. Antimicrob Agents Chemother. 2009 Jan;53 vancomycin or with resistance to metronidazole. Antimicrob Agents (1):261–263. PubMed PMID: 18955523; PubMed Central PMCID: Chemother. 2005 Mar;49(3):1157–1159. PubMed PMID: 15728918; PMCPMC2612159. PubMed Central PMCID: PMCPMC549223. 66. Orenstein R, Dubberke E, Hardi R, et al. Safety and Durability of 82. Pullman JPJ, Leach TS Ramoplanin versus vancomycin in the treatment RBX2660 (Microbiota Suspension) for Recurrent Clostridium difficile of Clostridium difficile diarrhea: a phase 2 study. 44th Intersci. Conf. Infection: results of the PUNCH CD Study. Clin Infect Dis. 2016 Mar Antimicrob. Agents Chemother. Washington, DC 2004. p. abstr. K-985a. 1;62(5):596–602. PubMed PMID: 26565008; eng. 83. Critchley IA, Green LS, Young CL, et al. Spectrum of activity and mode of • Early data from a new commercial single use enema package action of REP3123, a new antibiotic to treat Clostridium difficile for CDI infections. J Antimicrob Chemother. 2009 May;63(5):954–963. 67. Dubberke ER, Mullane KM, Gerding DN, et al. Clearance of PubMed PMID: 19258353. Vancomycin-Resistant Enterococcus Concomitant With Administration 84. Citron DM, Warren YA, Tyrrell KL, et al. Comparative in vitro activity of a Microbiota-Based Drug Targeted at Recurrent Clostridium difficile of REP3123 against Clostridium difficile and other anaerobic intest- Infection. Open Forum Infect Dis. 2016 Sep;3(3):ofw133. PubMed PMID: inal bacteria. J Antimicrob Chemother. 2009 May;63(5):972–976. 27703995; PubMed Central PMCID: PMCPMC5047394. PubMed PMID: 19240076. EXPERT OPINION ON INVESTIGATIONAL DRUGS 13

85. Ochsner UA, Bell SJ, O’Leary AL, et al. Inhibitory effect of REP3123 Nov/2018 Nov 8. Available from: https://www.fiercebiotech.com/bio on toxin and spore formation in Clostridium difficile, and in vivo tech/j-j-scraps-phase-3-antibiotic-program-acquired-30b-actelion- efficacy in a hamster gastrointestinal infection model. J Antimicrob takeover Chemother. 2009 May;63(5):964–971. PubMed PMID: 19251726. 95. Alam MZ, Wu X, Mascio C, et al. Mode of Action and Bactericidal 86. Nayak SU, Griffiss JM, Blumer J, et al. Safety, Tolerability, Systemic Properties of Surotomycin against Growing and Nongrowing Exposure, and Metabolism of CRS3123, a Methionyl-tRNA Clostridium difficile. Antimicrob Agents Chemother. 2015;59 Synthetase Inhibitor Developed for Treatment of Clostridium diffi- (9):5165–5170. cile, in a Phase 1 Study. Antimicrob Agents Chemother. 2017 96. Daley P, Louie T, Lutz JE, et al. Surotomycin versus vancomycin in Aug;61(8). PubMed PMID: 28584140; PubMed Central PMCID: adults with Clostridium difficile infection: primary clinical outcomes PMCPMC5527627. DOI:10.1128/AAC.02760-16. from the second pivotal, randomized, double-blind, Phase 3 trial. 87. Rashid MU, Dalhoff A, Backstrom T, et al. Ecological impact of J Antimicrob Chemother. 2017 Dec 1;72(12):3462–3470. PubMed MCB3837 on the normal human microbiota. Int J Antimicrob PMID: 28961905; eng. Agents. 2014 Aug;44(2):125–130. PubMed PMID: 24931053. 97. Boix V, Fedorak RN, Mullane KM, et al. Primary Outcomes From 88. Rashid MU, Dalhoff A, Weintraub A, et al. In vitro activity of a Phase 3, Randomized, Double-Blind, Active-Controlled Trial of MCB3681 against Clostridium difficile strains. Anaerobe. 2014 Surotomycin in Subjects With Clostridium difficile Infection. Aug;28:216–219. PubMed PMID: 25016084. Open Forum Infect Dis. 2017 Winter;4(1):ofw275. PubMed 89. Endres BT, Basseres E, Alam MJ, et al. Cadazolid for the treatment of PMID: 28480267; PubMed Central PMCID: PMCPMC5414029. Clostridium difficile. Expert Opin Investig Drugs. 2017 Apr;26 eng. (4):509–514. PubMed PMID: 28286992. 98. Magee G, Strauss ME, Thomas SM, et al. Impact of Clostridium 90. Gehin M, Desnica B, Dingemanse J. Minimal systemic and high difficile-associated diarrhea on acute care length of stay, hospital faecal exposure to cadazolid in patients with severe Clostridium costs, and readmission: A multicenter retrospective study of inpa- difficile infection. Int J Antimicrob Agents. 2015 Nov;46(5):576–581.. tients, 2009–2011. Am J Infect Control. 2015 Nov;43(11):1148–1153. PubMed PMID: 26419191. PubMed PMID: 26521932; eng. 91. Petrosillo N, Granata G, Cataldo MA. Novel Antimicrobials for the 99. Tabak YP, Zilberberg MD, Johannes RS, et al. Attributable burden of Treatment of Clostridium difficile Infection. Front Med (Lausanne). hospital-onset Clostridium difficile infection: a propensity score 2018;5:96. PubMed PMID: 29713630; PubMed Central PMCID: matching study. Infect Control Hosp Epidemiol. 2013 Jun;34 PMCPMC5911476. (6):588–596. PubMed PMID: 23651889; eng. 92.LouieT,NordCE,TalbotGH,etal. Multicenter, Double-Blind, 100. Lessa FC, Mu Y, Bamberg WM, et al. Burden of Clostridium difficile Randomized, Phase 2 Study Evaluating the Novel Antibiotic Infection in the United States. N Engl J Med. 2015;372(9):825–834. Cadazolid in Patients with Clostridium difficile Infection. PubMed PMID: 25714160. Antimicrob Agents Chemother. 2015 Oct;59(10):6266–6273. 101. Bouza E. Consequences of Clostridium difficile infection: under- PubMed PMID: 26248357; PubMed Central PMCID: PMCPM- standing the healthcare burden. Clin Microbiol Infect. 2012 C4576054. eng. Dec;18(Suppl 6):5–12.. PubMed PMID: 23121549; eng. 93. Gerding DN, Hecht DW, Louie T, et al. Susceptibility of Clostridium 102. Chopra T, Goldstein EJ, Gorbach SL. Rethinking Strategies to Select difficile isolates from a Phase 2 clinical trial of cadazolid and Antibiotic Therapy in Clostridium difficile infection. Pharmacotherapy. vancomycin in C. difficile infection. J Antimicrob Chemother. 2016 2016 Dec;36(12):1281–1289.. PubMed PMID: 27862113; eng. Jan;71(1):213–219. PubMed PMID: 26433782; PubMed Central 103. Aguado JM, Anttila VJ, Galperine T, et al. Highlighting clinical needs PMCID: PMCPMC4681371. eng. in Clostridium difficile infection: the views of European healthcare 94. Taylor NP Johnson & Johnson scraps phase 3 antibiotic program professionals at the front line. J Hosp Infect. 2015 Jun;90 acquired in $30B Actelion takeover: FierceBiotech; 2018 cited 2018 8/ (2):117–125. PubMed PMID: 25842241; eng.