ABSTRACT

Efficacy of CD101, a Novel Echinocandin, in Prevention of Pneumocystis Pneumonia (PCP): Thwarting the Biphasic Life Cycle of Pneumocystis

M.T. Cushion,1,2 A. Ashbaugh,1,2 K. Lynch,1,2 M.J. Linke,1,2 K. Bartizal31Cincinnati VAMC, Cincinnati, OH; 2Univ. of Cincinnati Coll. of Medicine, Cincinnati, OH; 3Cidara Therapeutics, San Diego, CA

Background: Pneumocystis spp. are the obligate pathogenic fungi that cause PCP in immunocompromised mammalian hosts. The life cycle of Pneumocystis is thought to be biphasic with asexual replication by trophic forms and sexual production leading to formation of asci/cysts. There is evidence to suggest that infection is initiated by cyst inhalation and asci formation may be essential for the Pneumocystislife cycle. We have previously reported (Cushion, et al. 2010; 5:e8524) that currently available echinocandins are not suitable for monotherapy as large numbers of trophic forms remained after 3 weeks of treatment and asci re-appeared and replication resumed after treatment discontinuation. We evaluated the effects of CD101, a novel long-acting echinocandin, on trophic and cyst forms of Pneumocystis murina and its efficacy in preventing infection in a mouse model of PCP.

Methods: Mice (C3H/HeN) were immunosuppressed throughout this 6-week study by dexamethasone (4 mg/L) in acidified drinking water. Infection with P. murina was by intranasal inoculation (2 x 106/50 µL). Mice were divided into groups to receive either vehicle (untreated control), trimethoprim/sulfamethoxazole (TMP/SMX 50/250 mg/kg x 3 per week), or CD101 (20, 2, or 0.2 mg/kg once or 3x per week) intraperitoneally at the time mice were inoculated. After 6 weeks, mice were humanely euthanized and lungs were processed for analysis by homogenization. Slides made from the lung homogenates were prepared for quantification of trophic forms by rapid Wright-Giemsa stain and of asci by cresyl echt violet staining. The reduction in P. murina burden (nuclei of trophic forms and asci) in treatment and untreated control groups was evaluated. The nuclei and asci counts for each lung were log transformed and analyzed by ANOVA. Individual groups were compared by Dunn's test for multiple comparisons (GraphPadPrismv.6).

Results: The reduction in nuclei and asci counts for all groups are shown in the Figure. Statistically significant reductions in nuclei levels compared with untreated controls were demonstrated with all CD101-treated groups except for the 0.2 mg/kg once weekly group. CD101 efficacy was comparable to that of TMP/SMX for 3 of the CD101 dose groups; no nuclei were observed by microscopic evaluation following CD101 dosages of 2 or 20 mg/kg 3x per week or 20 mg/kg once weekly. In terms of asci reduction, all CD101 groups demonstrated statistically significant reductions compared with untreated controls. CD101 efficacy was comparable to that of TMP/SMX for 5 of the CD101 dose groups (all but the 0.2 mg/kg once weekly group), with no asci observed by microscopic evaluation.

Conclusion: Asci/cysts appear to be critical for replication as well as transmission of Pneumocystis. CD101 inhibited the formation of both trophic forms and asci of P. murina in this mouse model of PCP in immunocompromised mice. These data demonstrate that CD101 is a viable candidate for prophylactic therapy of PCP.

• Pneumocystis spp. are detected in the lungs of most mammals. In immunocompromised hosts, they can cause lethal pneumonia (PCP) if untreated.

• C3H/HeN mice were immunosuppressed by dexamethasone in acidified drinking water and infected with P. murina by intranasal inoculation (2 x 106/50 µL).

• Mice were divided into 8 groups (n=10/group) to receive either vehicle (negative control), TMP/SMX, or CD101 administered intraperitoneally at the time of inoculation (Table).

• All but the 0.2 mg/kg CD101 dose given once per week significantly reduced total organism counts

• Three of the CD101 groups were not significantly different from the current standard treatment (TMP/SMX), with no organisms microscopically detected (20 mg/kg given once or 3x per wk and 2 mg/kg given 3x/wk)

• In previous studies, mice treated with anidulafungin and lacking detectable cysts could not transmit infection, suggesting the cyst is the agent for transmission (4).

• Therapeutic treatment with anidulafungin, caspofungin, or micafungin significantly reduced Pneumocystis cysts/asci burden but left large numbers of trophic forms. Cysts/asci gradually repopulated lungs upon cessation of echinocandin treatment (4).

• In the current study, CD101 significantly reduced total counts of trophic and asci forms compared with untreated controls in all but the 0.2 mg/kg 1x/wk dose group.

• Reduction of trophic nuclei with CD101 at higher doses were comparable to that of TMP/SMX. CD101 reduction of asci was also comparable in all but one group (0.2 mg/kg 1x/wk).

INTRODUCTION

ACKNOWLEDGMENTS / DISCLOSURESEditorial assistance was provided by Tressa Chung, RPh, CMPP (TMC Medical Communications) and was funded by Cidara Therapeutics. A.A., K.L., and M.J.L. have no relationships to disclose. M.C. is employed by Univ. of Cincinnati and has received research funding from Cincinnati VAMC and Cidara Therapeutics. K.B. is employed by and an equity owner of Cidara Therapeutics.

Immunosuppression (i.s.): Dexamethasone (4µg/L) in acidified drinking water (sulfuric acid; 1ml/L); i.s. continued through 6 wk

Extractlungs

Homogenize

Infection: Treatment:

Mince

2 x 106/50 µl, P. murina 10%DMSO/1% Tween/PBSIntra-nasally IP/mg/kg

(2d post-i.s.)

6 wk

Microscopic enumerationlog transformed, Kruskall-Wallis, Dunn’s post testfor multiple comparisons (GraphPadPrismv.6).

METHODS

Group 1 2 3 4 5 6 7 8

Drug Neg. control CD101 TMP/

SMX

Dose - 20 mg/kg

20 mg/kg

2 mg/kg

2 mg/kg

0.2 mg/kg

0.2 mg/kg

50/250 mg/kg

Freq. 3x/wk 1x/wk 3x/wk 1x/wk 3x/wk 1x/wk 3x/wk

• Mice were euthanized after 6 weeks, and lung homogenates were prepared for quantification of trophic and asci forms by rapid Wright-Giemsa and cresyl echt violet stains, respectively.

• The reduction in P. murina burden (nuclei of trophic forms and asci) in treated and untreated control groups was evaluated. The nuclei and asci counts for each lung were log transformed and analyzed by ANOVA. Individual groups were compared by Dunn's test for multiple comparisons (GraphPadPrismv.6).

RESULTS DISCUSSION

REFERENCES

• All CD101 groups caused a significant reduction in numbers of asci compared with the untreated controls

• There was no difference in efficacy between 5 of the CD101 groups (all but the 0.2 mg/kg x 1/wk) and TMP/SMX, with no organisms microscopically detected.

LOD = limit of detection; *P<0.05 vs control group.

1. Sandison T, et al. Safety and pharmacokinetics of CD101 IV in healthy adults. Antimicrob Agents Chemother. Submitted.

2. ClinialTrials.gov. RADIANT: CD101 vs standard of care in subjects with acute vaginal yeast infections. NCT02733432.

3. Ong V, et al. Subcutaneous (SC) injection of CD101, a novel echinocandin: efficacious, well-tolerated and sustained-release exposures. Presented at ICHS-Infocus, Santiago, Chile, 2016.

4. Cushion M, et al. Echinocandin treatment of Pneumocystis pneumonia in rodent models depletes cysts leaving trophic burdens that cannot transmit the infection. PLoS One. 2010;5:e8524.

CONCLUSIONS

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• Major forms of Pneumocystis are the smaller trophic form (asexual replication) and the larger asci/cyst form (sexual reproduction) that are thought to initiate infection when inhaled.

• Current standard treatment is trimethoprim/sulfamethoxazole (TMP/SMX), which targets folic acid synthesis. However, there is evidence of emerging resistance to both TMP and SMX, and some patients suffer severe allergic reactions and hepatotoxicity with treatment.

• Echinocandins target the fungal cell wall by inhibiting production of β-(1,3)-glucan, which is found in the cell wall of Pneumocystis asci/cyst form.

• CD101 is a novel echinocandin with long-acting pharmacokinetics being developed as a once-weekly, intravenous treatment of invasive fungal infections (1). The stability of CD101 enables additional dosage forms, such as subcutaneous and topical formulations, which are also in development (2,3).

• Here we report results from a 6-week study that evaluated the effects of CD101 on trophic and cyst forms of Pneumocystis murina and CD101 efficacy in preventing infection in a mouse model of PCP.

Mouse model of PCP prophylaxis

• Asci/cysts appear to be critical for replication as well as transmission of Pneumocystis infection.

• Treatment with CD101 resulted in statistically significant reductions in total numbers of P. murina vs untreated controls. In some dose groups, reductions in fungal burden with CD101 were comparable to those of TMP/SMX.

• CD101 blocked formation of both trophic and cyst/asci forms, suggesting that CD101 offers a potential new means to PCP prophylaxis.

LOD = limit of detection; *P<0.05 vs control group.

CD101 Trophic Nuclei Counts

Melanie T. Cushion, Ph.D.University of Cincinnati College of Medicine

231 Albert Sabin Way, ML 560Cincinnati, OH 45267

cushiomt@ucmail.uc.edu

ASH 20163396