1. My Home Town a Few Days Ago
I want to thank the organizers very much for the opportunity to participate again in this meeting which has become so successful. It has become one of our very best fungal meetings on an international basis. In preparing this discussion, I have tried to keep the title which contains the word relevance in mind throughout the discussion rather than try to display all the combination studies that have now been published. Therefore the studies I will show have been highly selected to make certain points regarding relevance. I apologize in advance to those of you who have done combination studies which I will not show here during the brief time we have for this discussion. Before getting into specific studies, I would like to reflect in a more general way with you on the general issue of combination therapy as it relates to fungal infections.

2. Relevance of Combination Antifungal Therapy: Some Key Questions
Is there a need for combination therapy in the first place?
Does the toxicity risk merit the use in attaining a “possible” mortality reduction?”
Are the doses of the investigational drugs appropriate?
Is the endpoint of the study meaningful?
I want to thank the organizers very much for the opportunity to participate again in this meeting which has become so successful. It has become one of our very best fungal meetings on an international basis. In preparing this discussion, I have tried to keep the title which contains the word relevance in mind throughout the discussion rather than try to display all the combination studies that have now been published. Therefore the studies I will show have been highly selected to make certain points regarding relevance. I apologize in advance to those of you who have done combination studies which I will not show here during the brief time we have for this discussion. Before getting into specific studies, I would like to reflect in a more general way with you on the general issue of combination therapy as it relates to fungal infections.

3. Practical Advantages of Antifungal Combination Therapy
From Kontoyiannis and Lewis
Synergy Broader Spectrum Decreased Resistance Pharmacokinetic Enhancement Better Tolerance with Lower Doses
The advantages of combination therapy have been enumerated here by Kontoyiannis and Lewis are are:

4. Combination Strategies Unique to Fungal Infections
Overall Mortality Rates Very High Toxicity of Antifungals is Significant Fungal Diseases Relatively Rare Drug Costs High Superiority Trials for Efficacy Preferred Costs of Studies Enormous
John Powers has written a most thoughtful editorial on the subject of combination antifungal therapy for fungi and I would recommend it to all interested in the general concepts. With his help, we can reflect on some of the issues related to combination theapy that are relatively unique to fungal infections. They are on the next two slides:
Considerations in Clinical Trials of Combination Antifungal Therapy: John H. Powers CID 39: S228: 2004

5. Unique Aspects of Combination Treatment for Fungal Infections
Surrogate Endpoints Rare/non-existent Multiple Companies Needed for Studies Historical Trials Problematic Relatively Few Agency Approved Drugs Standard of Care May Be Combinations
Considerations in Clinical Trials of Combination Antifungal Therapy: John H. Powers CID 39: S228: 2004

6. Relevance of Combination Antifungal Therapy: Some Key Questions
Is there a need for combination therapy in the first place?
Definite:
Aspergillosis
Mucormycosis
Fusarium, Scedoporium, and other moulds
Coccidiomycosis
Probable
Cryptococcosis
Candidiasis: Especially specific forms such
as endocarditis, osteomyelitis, and endophthalmitis
Now lets return to the four questions on our earlier slide regarding relevance.

7. Successfully Treated Candida krusei Infection of the Lumbar Spine with Combined Caspofungin/posaconazole Therapy: Schilling et al. Medical Mycology Jan 2007
While I did not include candida, an artifical classification, here is an example where combination therapy is still of need in regard to specific organ involvement. I fully recognize that many would still consider the mortality of candidemia too high with the single agents we currently have available. However, with the pressing need for further combination trials for aspergillus, I believe Candida will not get the same attention due to priority assignment by those designing clinical trials.

8. Relevance of Combination Antifungal Therapy: Some Key Questions
Does the toxicity risk merit the use in attaining a “possible” mortality reduction?”

9. Warfarin Cyclosporin Tacrolimus Sulfonureas Statins Benzodiazepines
Voriconazole Interactions
Vinca Alkaloids Sirolimus Rifabutin Rifampicin Terfenadine
Warfarin Cyclosporin Tacrolimus Sulfonureas Statins Benzodiazepines
Added toxicity is an issue we will always need to consider with antifungal combiantions. We are all familiar with the multiple toxicities that drugs in combination might have as exemplified by the numerous drug-drug interactions associated with voriconazole

10. Relevance of Combination Antifungal Therapy: Some Key Questions
Are the doses of the investigational drugs appropriate?

11. Synergism vs Cryptococcal Meningitis
N Engl J Med: 301:126, 1979
51 Courses, 10wks AMB Alone vs. 6 Weeks of Combo
Success %
67%
Combo
5-FC: 150mg/kg/d + Amb: 0.3 mg/kg/d
AMB: 0.4 mg/kg/d
41%
The issue of what dose to use when drugs are used in combination is always highly complex. This study, one of the first very carefully done combination studies is one in which the dose of amphotericin B, especially in the comparator arm has been considered by many as too low, and in fact is not the conventional dose which has evolved for the management of cryptococcal meningitis

12. Relevance of Combination Antifungal Therapy: Some Key Questions
Is the endpoint of the study meaningful?
Underlying Condition of Patient
All Cause Mortality
Fungal Related Mortality
Break Through Fungal Infection
Composite Endpoint
Microbiologic Cure
Clinical Cure
Time of the End Point Analysis
Surrogate Marker End Point
Cost of Care
Use of Alternative Strategies
Toxicity
Retrospective Subgroup Analysis
This point has always been the source for considerable discussion regarding the evaluation of any combination study for its relevance. Here are just some of the points which are raised regarding the relevance and significance of the study.

13. Relationship Between Severity of Disease at Baseline, As Measured by APACHE II, and Clinical Outcomes: From Rex et al. Clin Inf Dis: 36:1221, 2003
What about the issue of the underlying co-morbidities influencing the outcome of the study. Here we see from one of the early candidemia studies the important impact of the Apache II score on the outcome of two comparative arms. If one looks at the relatively well patients and the very ill patients there is no difference in success, but in the patients in the intermediate situation there is a significant difference in the two treatment arms.

14. Combination of Voriconazole and Caspofungin as Primary Therapy for Invasive Aspergillosis in Solid Organ Transplant Recipients: A Prospective, Multicenter, Observational Study Singh et al. Transplantation 81:320, 2006 P
Combination
Lipid Ampho Group
Days Post Diagnosis
Probability of Survival
Vori + Caspo
N=40
N=47
P=0.1
Vori + Caspo Mortality
Due to IA: 26%
LAMB Grp Mortality
Due to IA: 43%
In analysis of this study, the questions were asked of the authors what the underlying condition had on the outcome of combination therapy with vori and caspo in this prospective, observational study. They had done the analysis and found no effect of the underlying conditions.

15. Singh et al. to Bal Regarding Mortality Stratified by Disease Transplantation, Letter p291, 2006
Conclusion:
Condition of the Patients
did not Influence Outcome

16. Combination Therapy for Invasive Aspergillosis: Marr et al
Combination Therapy for Invasive Aspergillosis: Marr et al. : CID 39:737, 2004 (Retrospective, Salvage)
Vori + Caspo
n=31
n=16
P=.048

17. Combination Therapy for Invasive Aspergillosis: Marr et al
Combination Therapy for Invasive Aspergillosis: Marr et al. : CID 39:737, 2004

18. Survival After Combination Therapy for Aspergillosis: Marr et al
Survival After Combination Therapy for Aspergillosis: Marr et al. Clin Inf Dis 40:1074, 2005
Vori + Caspo
Voriconazole
Probability of Survival %
Days After Diagnosis
P=0.26
The benefit of the combination treatment seen at 90 days
was not present when the survival at a year was analyzed.

19. Marr in Response to Cesaro and Visintin: CID 40:1075, 2005
Probability of Survival
Death Due To IA
Death From Other
Vori
Combo
Vori + Caspo *
Marr in Response to Cesaro and Visintin: CID 40:1075, 2005
One year data related to causes of death

20. High-dose Caspofungin (100mg/d) Combination Antifungal Therapy in Patients with Hematologic Malignancies and Hematopoietic Stem Cell Transplantation: Safdar et al. Bone Marrow Trans: 39:157:2007 (Retrospective)
All Cause Mortality
Caspo + OLAT
N=31
P=0.1
N=63
*More immunostim

21. Invasive Aspergillosis Following Hematopoietic Cell Transplantation: Outcomes and Prognostic Factors Associated with Mortality: Upton et al. CID 44:531: 2007 (Retrospective, 405 Patients)
Probability of Attributable Death in Patients With IA according to Years
Year of Diagnosis
This recently published study shows the potential hazzard of using historical controls as a comparator group. It demonstrates an improvement in survival related to time. Each line is a two year period dating from 1990 to The curve keeps improving with regard to the probability of survival.

22. No. of Days After IA Diagnosis
Invasive Aspergillosis Following Hematopoietic Cell Transplantation: Outcomes and Prognostic Factors Associated with Mortality: Upton et al. CID 44:531: 2007 (Retrospecive Analysis of 405 Patients)
Probability of Attributable Death in Patients Receiving and not Receiving Voriconazole
Other
Voriconaozle
Probability of Death Due to IA
No. of Days After IA Diagnosis
P=.03
N=54
N=176
The population which received voriconazole had a lower probability of death due to invasive aspergillosis,compared to the population receiving regimens not containing vori

23. Reasons for Improved Survival Over Time:
Invasive Aspergillosis Following Hematopoietic Cell Transplantation: Outcomes and Prognostic Factors Associated with Mortality: Upton et al. CID 44:531: 2007 (Retrospective Analysis of 405 Patients)
Reasons for Improved Survival Over Time:
Change in Transplant Practices
Non-myeloablative Suppression
Stem Cell Transplantation
Improved Diagnosis of Aspergillosis
Use of Voriconazole
Interpreting Historical Controls May Be Highly Complex
These are the following reasons for the improved survival.

24. Selected Combination Studies for Aspergillosis

25. Chemotherapy for Acute Leukemia n=20 75% Response
Refractory Fungal Pneumonia in Patients with Acute Leukemia: Successful Treatment with Combination Caspofungin and Amphotericin B: Aliff et al. Cancer 97:1025, 2003 (Retrospective, Salvage Study)
Proven, Probable, and Possible (Most Possible)
30 Patients Total: Retrospective Study
Response=Improvement
All Patients n=30
60% Response
Chemotherapy for Acute Leukemia n=20
75% Response
Survival at Discharge Higher with Patients Having
a Favorable Response

26. Multicenter, Noncomparative Study of Caspofungin in Combination With Other Antifungals as Salvage Therapy in Adults With Invasive Aspergillosis. Maertens et al. Cancer 107:2888, 2006 (Open Label, Non-Comparative, Refractory or Intolerant)

27. High Dose Ambisome vs. Ambisome + Caspo
For Aspergillosis: Caillot et al. Cancer, Oct. 16, 2007
15 Patients Each Arm: High Dose AMB 10mg/kg, Standard 3mg/kg
Survival at Week 12: High Dose Ambisome 80% Combo 100%

28. Combination Salvage Therapy of Invasive Aspergillosis in Patients with Hematologic Malignancy: Which Caspofungin-Containing Regimen?
Raad et al. 47th ICAAC Abstract M-624, 2007 (Retrospective, Salvage Study)
Vori +
Caspo
Res
Response
Rate
All Cause
Mortality
IA Mortality
Renal
Toxicity
Percent *
*: Sig
LP-AMB + 59 33

29. Raad et al. 47th ICAAC Abstract M-624, 2007 (Salvage Study)
Combination Salvage Therapy of Invasive Aspergillosis in Patients with Hematologic Malignancy: Which Caspofungin-Containing Regimen?
Raad et al. 47th ICAAC Abstract M-624, (Salvage Study)
GCSF Or
Other
Number of
Patients
Patient
age
Acute
Leukemia 59 52
48%
85% 33 58
12%
64%
Caspo +LPAMB
Caspo + Vor
All Statistically Significant

30. Adding Rifampicin to Caspofungin may show some additive effects:
In Vitro Synergy Testing of Rifampicin with Caspofungin and Amphotericin B against Aspergillus spp. And Fusarium spp.: Odabasi et al.: 47th ICAAC, Abstract M-546, 2007
Conclusions:
Combination of Rifampicin with Amphotericin B seems very active against commonly seen molds:
Adding Rifampicin to Caspofungin may show some additive effects:
Amphotericin B and Rifampicin combinations should be further tested with in-vivo studies

31. Single Agent or Combination to Treat Invasive Aspergillosis?
Kubin et al. 46th ICAAC, Abstract M-899, 2006
Retrospective 146 proven/probable primary cases
Mono
N=124
47 AMB: 33 Vori
Caspo + Vori
N=22
Response
24%
21%
12 Week Mortality
55%
46%

32. Retrospective, 238 Proven/Probable Cases with Hematological Malignancy
Caspofungin Plus Posaconazole vs. Liposomal Amphotericin B for Aspergillosis: Raad et al. 45th ICAAC, Abstract M-1035, 2004
LAMB + Caspo
N=48
Posa + Caspo
N=43
14%
29%
24%
32%
Response
Excluding ICU
Survival at Discharge Higher with Posa Combination Therapy

33. Antifungal Interactions with the Triple Combination of Amphotericin B, Caspofungin, and Voriconazole Against Aspergillus Species
Syngergism at Low Concentrations
AMB mg/L
Vori mg/L
Caspo mg/L
Antagonism at High Concentrations
AMB mg/L
Vori mg/L
Caspo Same Range
O’Shaughnessy et al. J ANtimicrob Chemother 58:1168, 2006

34. Micafungin, Alone or in Combination with Other Systemic Antifungal Agents, for the Treatment of Acute Invasive Aspergillosis: Denning et al. J of Infect, 53:337, 2006 (Open Label, Non-Comparative, Prospective)
Percent Survival
Primary
Salvage
225 Patients
6/12
5/17
9/22
60/174
98/225 HSCT: 88/98 /allo
48 GVHD
83/225 Received Chemo
For Malignancy

35. Micafungin, Alone or in Combination Against Aspergillus
Kontoyiannis et al. 46th ICAAC, Abstract M-878, 2006
Refractory in Bone Marrow Transplant Patients
N=8
N=90
Response %
38%
24 %

36. Combination Strategies for Non-Aspergillus Fungi

37. Pappas et al. 47th ICAAC Abstract M-626, 2007
Fluconazole Plus Amphotericin B vs AMB Alone for Primary Treatment of AIDS-Associated Cryptococcal Meningitis; Results of a Phase II Trial
Pappas et al. 47th ICAAC Abstract M-626, 2007
Standard Therapy: 0.7 mg/kg AMB for 14 days then 8 weks of 400 mg Flu
Low Dose: AMB plus 400mg of Flu for 14 days then 400 mg Flu for 8weeks
High Dose: AMB plus 800mg of Flu for 14 days ten 800 mg Flu for 8 weeks
Success for End Point: CSF Cultures neg, neurological stability,
and survival at day 14

38. Pappas et al. 47th ICAAC Abstract M-626, 2007
Fluconazole Plus Amphotericin B vs AMB Alone for Primary Treatment of AIDS-Associated Cryptococcal Meningitis; Results of a Phase II Trial
Pappas et al. 47th ICAAC Abstract M-626, 2007
Standard Rx
Low Dose
High Dose
N=46
% Success: 41
N=42
% Success: 31
% Success: 55
N=37
% Success: 76
N=35
% Success: 80
N=33
% Success: 85
N=38
% Success:76
N=32
% Success:88
N=28
% Success:93
Day 14
Day 42
Day 70

39. Activity of Caspofungin Alone and in Combination with Amphotericin B Lipid Complex in a Murine Model of Fusariosis: Ostrosky-Zeichner et al. 47th ICAAC Abstract M-1841, 2007
Placebo
Caspo
10mg/kg
ABLC
Combo
10mg
5mg
Percent Survival
25 Mice Per Group *

40. Synergism of L-AMB and Micafungin Combination in Murine Mucormycosis: Spellberg et at. 46th ICAAC, Abstract M-1744, 2006
Survival %

41. Reed et al. 45th IDSA, Abstract 659, 2007
Combination Polyene-Echinocandin Therapy in the Treatment of Mucormycosis
Reed et al. 45th IDSA, Abstract 659, 2007
10 Year Retrospective Review
Endpoint: Survival for 30 Days Following Discharge
10 Patients Found: All rhinocerebral (9/10) CNS Involvement
Combination echinocandin + Polyene: 3/3 survivors
Polyene alone: /7 Survivors
Conclusion:
Prospective Investigation of combination Polyene-
Echinocandin Therapy for the Treatment of Mucormycosis
is Warranted

42. The Iron Chelator Deferasirox Protects Mice From Mucormycosis Through Iron Starvation:
Ibrahim et al. JCI 117:2649: 2007
Desferoxamine
Placebo
Deferasirox

43. Chelator Combined with L-AMB
The Iron Chelator Deferasirox Protects Mice From Mucormycosis Through Iron Starvation:
Ibrahim et al. JCI 117:2649: 2007
Deferasirox
Chelator Combined with L-AMB
Placebo
Desferoxamine

44. Summary: Relevance of Combination Therapy
Majority of existing studies are anecdotal, retrospective,
and or non-comparative
Prospective, double blind studies are exceedingly problematic
in design issues and feasibility
Prospective, double blind, trials will be forthcoming
but highly restricted in number
Weight of the evidence is in favor of combination therapy
in seriously ill patients with invasive fungal infections
Until studies are completed, use combination therapy in
serious cases
Tolerance of the patient for the combination needs to be
carefully monitored to justify the use.

45. Grazie!