U.S. patent application number 15/655372 was filed with the patent office on 2017-11-16 for compositions and methods for transplantation of colon microbiota.
This patent application is currently assigned to Regents of the University of Minnesota. The applicant listed for this patent is Regents of the University of Minnesota. Invention is credited to Matthew J. HAMILTON, Alexander KHORUTS, Michael J. SADOWSKY, Alexa R. WEINGARDEN.
Application Number | 20170326181 15/655372 |
Document ID | / |
Family ID | 46798573 |
Filed Date | 2017-11-16 |
United States Patent
Application |
20170326181 |
Kind Code |
A1 |
SADOWSKY; Michael J. ; et
al. |
November 16, 2017 |
Compositions and Methods for Transplantation of Colon
Microbiota
Abstract
The present invention provides compositions that include an
extract of human feces, and methods for using such compositions,
including methods for replacing or supplementing or modifying a
subject's colon microbiota, and methods for treating a disease,
pathological condition, and/or iatrogenic condition of the
colon.
Inventors: |
SADOWSKY; Michael J.;
(Roseville, MN) ; KHORUTS; Alexander; (Golden
Valley, MN) ; WEINGARDEN; Alexa R.; (St. Paul,
MN) ; HAMILTON; Matthew J.; (Burnsville, MN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Regents of the University of Minnesota |
Minenapolis |
MN |
US |
|
|
Assignee: |
Regents of the University of
Minnesota
Minenapolis
MN
|
Family ID: |
46798573 |
Appl. No.: |
15/655372 |
Filed: |
July 20, 2017 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15626880 |
Jun 19, 2017 |
|
|
|
15655372 |
|
|
|
|
15594087 |
May 12, 2017 |
|
|
|
15626880 |
|
|
|
|
15261319 |
Sep 9, 2016 |
9649343 |
|
|
15594087 |
|
|
|
|
14003411 |
Jan 17, 2014 |
|
|
|
PCT/US2012/028484 |
Mar 9, 2012 |
|
|
|
15261319 |
|
|
|
|
15173134 |
Jun 3, 2016 |
|
|
|
15594087 |
|
|
|
|
14003411 |
Jan 17, 2014 |
|
|
|
PCT/US2012/028484 |
Mar 9, 2012 |
|
|
|
15173134 |
|
|
|
|
15594319 |
May 12, 2017 |
|
|
|
15626880 |
|
|
|
|
15261319 |
Sep 9, 2016 |
9649343 |
|
|
15594319 |
|
|
|
|
14003411 |
Jan 17, 2014 |
|
|
|
PCT/US2012/028484 |
Mar 9, 2012 |
|
|
|
15261319 |
|
|
|
|
15173134 |
Jun 3, 2016 |
|
|
|
15594319 |
|
|
|
|
14003411 |
Jan 17, 2014 |
|
|
|
PCT/US2012/028484 |
Mar 9, 2012 |
|
|
|
15173134 |
|
|
|
|
15626898 |
Jun 19, 2017 |
|
|
|
14003411 |
|
|
|
|
15594087 |
May 12, 2017 |
|
|
|
15626898 |
|
|
|
|
15261319 |
Sep 9, 2016 |
9649343 |
|
|
15594087 |
|
|
|
|
14003411 |
Jan 17, 2014 |
|
|
|
PCT/US2012/028484 |
Mar 9, 2012 |
|
|
|
15261319 |
|
|
|
|
15173134 |
Jun 3, 2016 |
|
|
|
15594087 |
|
|
|
|
14003411 |
Jan 17, 2014 |
|
|
|
PCT/US2012/028484 |
Mar 9, 2012 |
|
|
|
15173134 |
|
|
|
|
15594319 |
May 12, 2017 |
|
|
|
15626898 |
|
|
|
|
15261319 |
Sep 9, 2016 |
9649343 |
|
|
15594319 |
|
|
|
|
14003411 |
Jan 17, 2014 |
|
|
|
PCT/US2012/028484 |
Mar 9, 2012 |
|
|
|
15261319 |
|
|
|
|
15173134 |
Jun 3, 2016 |
|
|
|
15594319 |
|
|
|
|
14003411 |
Jan 17, 2014 |
|
|
|
PCT/US2012/028484 |
Mar 9, 2012 |
|
|
|
15173134 |
|
|
|
|
61450838 |
Mar 9, 2011 |
|
|
|
61450838 |
Mar 9, 2011 |
|
|
|
61450838 |
Mar 9, 2011 |
|
|
|
61450838 |
Mar 9, 2011 |
|
|
|
61450838 |
Mar 9, 2011 |
|
|
|
61450838 |
Mar 9, 2011 |
|
|
|
61450838 |
Mar 9, 2011 |
|
|
|
61450838 |
Mar 9, 2011 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/1617 20130101;
Y02A 50/473 20180101; A61K 9/1623 20130101; A61P 31/04 20180101;
A61K 47/26 20130101; Y02A 50/475 20180101; A61K 35/24 20130101;
A61K 9/16 20130101; A61P 1/10 20180101; A61K 9/0053 20130101; A61K
35/74 20130101; A61K 35/741 20130101; A61P 1/12 20180101; A61P 1/04
20180101; Y02A 50/30 20180101; A61K 47/08 20130101; A61K 35/37
20130101; A61K 35/742 20130101; A61K 2035/11 20130101; A61P 1/00
20180101; A61K 45/06 20130101; A61K 35/74 20130101; A61K 2300/00
20130101; A61K 35/24 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 35/37 20060101
A61K035/37; A61K 47/26 20060101 A61K047/26; A61K 47/08 20060101
A61K047/08; A61K 35/742 20060101 A61K035/742; A61K 9/00 20060101
A61K009/00; A61K 35/24 20060101 A61K035/24; A61K 9/16 20060101
A61K009/16; A61K 9/16 20060101 A61K009/16; A61K 9/16 20060101
A61K009/16; A61K 35/74 20060101 A61K035/74; A61K 35/741 20060101
A61K035/741 |
Goverment Interests
GOVERNMENT FUNDING
[0002] This invention was made with government support under
R21A1091907, awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1-42. (canceled)
43. A method for treating a Clostridium difficile infection (CDI)
in a subject in need thereof, said method comprising administering
to said subject an effective amount of fecal microbe material that
is frozen for at least once, wherein said fecal microbe material is
capable of achieving a CDI clearance rate substantially similar to
a CDI clearance rate achievable by fresh fecal material not
previously frozen.
44. The method of claim 43, wherein said fecal microbe material is
subject to at least one freeze-thaw cycle.
45. The method of claim 43, wherein CDI clearance is defined as CDI
symptom resolution and negative PCR testing for C. difficile toxin
B after two months post said administering.
46. The method of claim 43, wherein said frozen is at a temperature
of -20.degree. C. or lower.
47. The method of claim 43, wherein the most abundant phylum of
said subject's post-administering fecal microbial community
structure is the phylum Firmicutes.
48. The method of claim 43, wherein said subject has had at least
one prior antibiotic treatment failure for said CDI.
49. The method of claim 43, wherein said suitable method is orally
administering to said subject.
50. The method of claim 43, wherein CDI clearance is identified by
resolution of diarrhea.
51. The method of claim 43, wherein said CDI clearance rate is
determined 2 months post said administering to said subject an
effective amount of said fecal microbe material.
52. The method of claim 43, wherein said fecal microbe material
consists essentially of particles capable of passing through a 0.5
mm sieve and an intestinal microbiota from a donor.
53. The method of claim 43, wherein said fecal microbe material
comprises an extract of feces from said donor.
54. The method of claim 43, wherein said fecal microbe material
comprises at least 5.times.10.sup.10 cells.
55. The method of claim 43, wherein said subject has an
inflammatory bowel disease prior to said administering.
56. The method of claim 43, wherein said subject is free of a
recurrent Clostridium difficile infection for at least two months
after said administering.
57. The method of claim 56, wherein said subject tests negative for
Clostridium difficile toxin B for at least two months after said
administering.
58. The method of claim 56, wherein said subject's diarrhea is
resolved for at least two months after said administering.
59. The method of claim 56, wherein said subject is free of a
recurrent Clostridium difficile infection for at least three months
after said administering.
60. The method of claim 43, further comprising pretreating said
patient with one or more antibiotics prior to said
administering.
61. The method of claim 60, wherein said one or more antibiotics
are selected from the group consisting of Metroidazole, Rifaximin,
Vancomycin, and Neomycin.
62. A method for treating a Clostridium difficile infection (CDI)
in a subject in need thereof, said method comprising administering
to said subject an effective amount of a previously frozen fecal
microbe material, wherein said fecal microbe material is capable of
achieving a CDI clearance rate substantially similar to a CDI
clearance rate achievable by fresh fecal material not previously
frozen, wherein CDI clearance is defined as CDI symptom resolution
and negative PCR testing for C. difficile toxin B after two months
post said administering.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/261,319 filed Sep. 9, 2016, which is a continuation of U.S.
application Ser. No. 14/003,411 filed Jan. 17, 2014, which is a 371
of International Application No. PCT/US2012/028484 filed Mar. 9,
2012, which claims the benefit of U.S. Provisional Application No.
61/450,838 filed Mar. 9, 2011, the entireties of each of which are
herein incorporated by reference. This application is also a
continuation of U.S. application Ser. No. 15/173,134 filed Jun. 3,
2016, which is a continuation of U.S. application Ser. No.
14/003,411 filed Jan. 17, 2014, which is a 371 of International
Application No. PCT/US2012/028484 filed Mar. 9, 2012, which claims
the benefit of U.S. Provisional Application Ser. No. 61/450,838,
filed Mar. 9, 2011, the entireties of each of which are herein
incorporated by reference.
BACKGROUND
[0003] In 1978, Clostridium difficile was first recognized as a
major cause of diarrhea and pseudomembranous colitis associated
with the use of antimicrobial agents. Since this time, infection by
C. difficile has been steadily growing in incidence, morbidity, and
mortality across North America and Europe (Freeman et al. Clin
Microbiol Rev 2010; 23:529-49; Kelly and LaMont, N Engl J Med 2008;
359:1932-40). Analysis of the U.S. National Hospital Discharge
Survey statistics between 1996 and 2003 reveals a doubling in the
prevalence of diagnosis of C. difficile infection (CDI), to
0.61/1,000, among inpatients (McDonald et al. Emerg Infect Dis
2006; 12:409-15). A 2008 survey of 12.5% of all U.S. acute care
facilities indicated a CDI prevalence rate of 13.1/1,000, which is
at least an order of magnitude higher than that found previously
(Jarvis et al. Am J Infect Control 2009; 37:263-70). While older
patients have disproportionately greater rates of CDI than younger
individuals, no age group is spared, and the incidence of
CDI-related hospitalizations has been rising even in the pediatric
population (Zilberberg et al. Emerg Infect Dis 2010; 16:604-9). The
increase in incidence has been further compounded by an elevated
frequency of the most severe forms of this disease, as evidenced by
rising CDI-associated morbidity and case fatality (Ricciardi et al.
Arch Surg 2007; 142:624-31; discussion 631, Zilberberg et al. Emerg
Infect Dis 2008; 14:929-31). This is, in part, related to the
emergence of more virulent C. difficile strains, such as PCR
ribotype 027/North American Pulsed Field type 1 (NAP1), which is
characterized by a greater potential for toxin production and
antibiotic resistance than other clinically-relevant strains
(Rupnik et al. Nat Rev Microbiol 2009; 7:526-36, Kuijper et al.
Euro Surveill 2008; 13).
[0004] Recurrent CDI is one of the most difficult and increasingly
common challenges associated with CDI (Surawicz, Gastroenterology
2009; 136:1152-4). An initial incidence of CDI can be followed by a
relapse within 30 days in about 20-30% of cases (Kelly and LaMont,
N Engl J Med 2008; 359:1932-40 (2008); Louie et al. N Engl J Med
2011; 364:422-31; Pepin et al. Clin Infect Dis 2006; 42:758-64),
and the risk of recurrence doubles after two or more occurrences
(McDonald et al. Emerg Infect Dis 2006; 12:409-15). Older age,
intercurrent antibiotic use for non-C. difficile indications, renal
insufficiency, immune deficiency, and antacid medications, are some
of the known risk factors for recurrent CDI (Surawicz,
Gastroenterology 2009; 136:1152-4, Garey et al. J Hosp Infect 2008;
70:298-304). The presence of just three clinical criteria: age
>65 years, severe disease, and continued use of antibiotics
after treating the initial CDI episode, are predictive of an almost
90% relapse rate (Hu et al. Gastroenterology 2009; 136:1206-14).
CDI also commonly complicates management of inflammatory bowel
disease (IBD), which has recently been recognized as an additional
independent risk factor for CDI infection (Issa et al. Clin
Gastroenterol Hepatol 2007; 5:345-51, Rodemann et al. Clin
Gastroenterol Hepatol 2007; 5:339-4415). CDI in patients with
underlying IBD is associated with increased severity of colitis and
higher rates of recurrence and colectomy (Issa et al. Inflamm Bowel
Dis 2008; 14:1432-42).
[0005] It is now recognized that the presence of normal, healthy,
intestinal microbiota (normal gut microorganisms) offers protection
against CDI. Conversely, severe disruption of normal intestinal
microbiota by use of antibiotics, including metronidazole and
vancomycin that are used to treat CDI, is likely one of the major
reason for its recurrence. Chang and colleagues used 16S rDNA
sequencing to analyze the fecal microbiota of seven patients with
initial and recurrent CDI (Chang et al. J Infect Dis 2008;
197:435-8). They reported that bacterial species diversity was
reduced in all patients compared to normal control subjects. The
greatest reduction in species diversity, however, was found in the
three patients with recurrent CDI and disruption of their gut
microbiota was evident at the phylum level--with marked reduction
in Bacteroidetes, normally one of the two dominant phyla in the
colon. Instead, the gut microbiota in these patients were dominated
by members of the proteobacteria and verrucomicrobia phyla, which
normally are only minor constituents of the colon microbiota.
[0006] The general aim of antibiotic treatment for recurrent CDI is
not mere suppression of C. difficile, but also preservation of the
residual colon microbiota and optimization of their restoration.
Various antibiotic regimens, including long tapered or pulsed
dosing with vancomycin (McFarland et al., Am J Gastroenterol 2002;
97:1769-75) and rifaximin "chaser" protocols (Johnson et al. Clin
Infect Dis 2007; 44:846-8, Johnson et al. Anaerobe 2009; 15:290-1)
have been used to achieve this objective with partial success.
Recently, fidaxomicin, a new macrocyclic antibiotic which is narrow
in spectrum and spares Bacteroides species, was shown to reduce the
initial relapse rate of CDI by 50% compared to vancomycin treatment
(Louie et al. N Engl J Med 2011; 364:422-31). However, treatment
with fidaxomicin did not alter the recurrence rate of CDI caused by
the more virulent PCR 027/NAP1 strain. Therefore, despite these
advances it seems likely that the challenges in treatment of
recurrent CDI will remain for the foreseeable future.
[0007] Fecal microbiota transplantation (FMT), also commonly known
as `fecal bacteriotherapy` represents the one therapeutic protocol
that allows the fastest reconstitution of a normal composition of
colon microbial communities. For many decades, FMT has been offered
by select centers across the world, typically as an option of last
resort for patients with recurrent CDI. The mostly commonly
earliest cited report for FMT was by Eiseman and colleagues who in
1958 described the use of fecal enemas for patients who likely had
severe or fulminant form of pseudomembranous colitis (Eiseman et
al. Surgery 1958; 44:854-9). Since this time, well over 200 cases
have been reported as individual case reports, or small case
series, with a .about.90% cumulative success rate in clearing
recurrent CDI, without any noted adverse events. The history and
general methodology used for FMT have been described in several
recent reviews (Bakken. Anaerobe 2009; 15:285-9, van Nood et al.
Euro Surveill 2009; 14, Khoruts and Sadowsky. Mucosal Immunol 2011;
4:4-7). However, despite the long and successful track record, as
well as great clinical need, the availability of the procedure for
many patients remains very limited.
[0008] The lack of wider practice of FMT is due in large part to
multiple non-trivial practical barriers and not due to lack of
efficacy. These include lack of reimbursement for donor screening,
lack of adequate donors at the correct time, difficulty in material
preparation and administration, as well as aesthetic concerns about
doing the procedure in endoscopy or medical office. These also
include patient perception of the procedure, willingness of staff
to perform the procedure, sanitation issues related to manipulation
of fecal matter. Together these factors make it a distasteful
option that is often considered a treatment of last resort, and
that is largely unavailable to the vast majority of patients who
could benefit from it. Moreover, the pharmaceutical industry has
shown little interest in technological development of FMT-based
therapeutics, in large part due to the wide availability of donor
material and its complex composition. Instead, development has been
driven mostly by individual clinicians faced with desperate need in
their patients.
SUMMARY OF THE INVENTION
[0009] The present invention provides compositions that include an
extract or a preparation of human feces. In one embodiment, a
composition includes no greater than 0.05%, 0.1%, 0.2%, 0.3%, 0.4%,
0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,
or 10% weight non-living material/weight biological material.
Optionally the biological material includes human gut, colon or
intestinal fecal microbes, and optionally the biological material
includes human gut, colon or intestinal bacteria. Optionally the
composition includes a pharmaceutically acceptable carrier.
Optionally the composition is a formulation for oral
administration.
[0010] In one embodiment, a composition consists of, or consists
essentially of, particles of non-living material and/or particles
of biological material that will pass through a sieve having a
sieve size of 2.0 mm, 1.0 mm, 0.5 mm, 0.25 mm, 0.212 mm, 0.180 mm,
0.150 mm, 0.125 mm, 0.106 mm, 0.090 mm, 0.075 mm, 0.063 mm, 0.053
mm, 0.045 mm, 0.038 mm, 0.032 mm, 0.025 mm, 0.020 mm, 0.01 mm, or
0.2 mm. Optionally the composition includes a pharmaceutically
acceptable carrier, and optionally the composition is a formulation
for oral administration.
[0011] In one embodiment, a composition includes at least 4
different phyla of gut, colon or intestinal bacteria extracted or
prepared from the gut, colon or intestine, wherein the phyla
include a member of Bacteroidetes phylum, member of Firmicutes
phylum, member of Proteobacteria phylum, member of Tenericutes
phylum, or a combination thereof. Optionally the phyla are chosen
from Bacteroidetes, Firmicutes, Proteobacteria, and Tenericutes.
The composition includes no greater than 0.05%, 0.1%, 0.2%, 0.3%,
0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,
9%, or 10% weight non-living material/weight biological material.
Optionally the biological material includes human gut, colon or
intestinal flora. Optionally the biological material includes human
gut, colon or intestinal bacteria. Optionally the composition
includes a pharmaceutically acceptable carrier, and optionally the
composition is a formulation for oral administration.
[0012] In one embodiment, a composition includes an extract of
human feces wherein the composition is substantially odorless,
optionally includes biological material, and optionally wherein the
biological material includes bacteria. Optionally the composition
includes a pharmaceutically acceptable carrier, and optionally the
composition is a formulation for oral administration.
[0013] A composition of the present invention may include no
greater than 0.1% weight non-living material/weight biological
material. In one embodiment, a composition may consist of, or
consist essentially of, particles that will pass through a 0.25 mm
sieve, or equivalent. In one embodiment, a composition may include
no greater than 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%,
0.8%, 0.9%, 1%, 2%, 3%, 4% 5%, 6%, 7%, 8%, 9% or 10% weight
non-living material/weight biological material. A composition of
the present invention may further include a cryoprotectant, such as
glycerol. In one embodiment, a composition may be at a temperature
of less than 0.degree. C. In one embodiment, a composition is a
solid, such as a powder. A composition of the present invention may
include at least 1.times.10.sup.10, 2.times.10.sup.10,
3.times.10.sup.10, 4.times.10.sup.10, or 5.times.10.sup.10
bacteria. In one embodiment, the biological material of a
composition may include a plurality of prokaryotic cells,
eukaryotic cells, or viruses; or a population of prokaryotic cells,
eukaryotic cells, and viruses, that is substantially identical to
or representative of or equivalent to a population of prokaryotic
cells, eukaryotic cells, and viruses present in a feces of a normal
healthy human. In one embodiment, the biological material of a
composition may include a population of prokaryotic cells and
viruses that is substantially identical to or representative of or
equivalent to a population of prokaryotic cells and viruses present
in the feces of a normal healthy human. In one embodiment, the
biological material of a composition includes a population of
prokaryotic cells, eukaryotic cells, or viruses that is
substantially identical to or representative of or equivalent to a
population of prokaryotic cells, eukaryotic cells, and viruses
present in the feces of a normal healthy human.
[0014] The present invention also provides composition prepared by
a process. In one embodiment, a process includes subjecting a fecal
sample to a condition or conditions that remove at least 91%, 92%,
93%, 94% 95%, 96%, 97%, 98%, 99% or more of the non-living material
present in the fecal sample. In one embodiment, a process includes
filtering a fecal sample with a filter medium, wherein the filter
medium includes a sieve size of no greater than 2.0 nun, 1.0 mm,
0.5 mm, 0.25 mm, 0.212 mm, 0.180 mm, 0.150 mm, 0.125 mm, 0.106 mm,
0.090 mm, 0.075 mm, 0.063 mm, 0.053 mm, 0.045 mm, 0.038 mm, 0.032
mm, 0.025 mm, 0.020 mm, 0.01 mm, or 0.2 mm to result in or to
generate a filtrate. Optionally a composition includes a biological
material, and optionally the biological material includes bacteria.
Optionally a composition includes a pharmaceutically acceptable
carrier. Optionally a composition is a formulation for oral
administration. Optionally the process may occur at a temperature
of no greater than 26.degree. C., 27.degree. C., 28.degree. C.,
29.degree. C., 30.degree. C., 31.degree. C., 32.degree. C.,
33.degree. C., or 34.degree. C.
[0015] The composition may include at least 4 different phyla of
bacteria, wherein the include a member of Bacteroidetes phylum,
member of Firmicutes phylum, member of Proteobacteria phylum,
member of Tenericutes phylum, or a combination thereof. Optionally
the phyla are chosen from Bacteroidetes, Firmicutes,
Proteobacteria, and Tenericutes. In one embodiment, the composition
further includes at least 5, 6, 7, 8, 9, or 10 different classes of
bacteria chosen from Actinobacteria, Bacteroidia, Bacilli,
Clostridia, Erysipelotrichi, Alphaproteobacteria,
Betaproteobacteria, Gammaproteobacteria, Mollicutes, and
Verrucomicrobiae.
[0016] The process may further include adding a cryoprotectant, for
instance, glycerol, to the composition. The process may further
include freezing the composition. The composition may be for use as
a therapeutic agent, and it may be for use in the treatment of a
disease or a pathological or iatrogenic condition of the colon. The
disease may be a disease or condition characterized by a
dysfunctional or pathological composition of colon microbiota, for
instance, a Clostridium difficile colitis.
[0017] The present invention also provides a method for replacing
or supplementing or modifying a subject's colon microbiota. The
method may include administering to the subject a composition
described herein. The present invention also provides a method for
treating a subject. The method may include administering to a
subject in need thereof an effective amount of a composition
described herein. The methods may further include removal of some,
most, or substantially all of the subject's colon, gut or
intestinal microbiota prior to the administering. The subject may
have or be at risk for having a colitis. In one embodiment, the
colitis is an autoimmune colitis, such as an inflammatory bowel
disease, an ulcerative colitis, a Crohn's disease, or an irritable
bowel syndrome. In one embodiment, the colitis is an infectious
colitis, such as a Clostridium difficile colitis or an
enterohemorrhagic colitis. The Clostridium difficile colitis may be
an acute Clostridium difficile colitis, a relapsing Clostridium
difficile colitis, and a severe Clostridium difficile colitis. The
enterohemorrhagic colitis may be caused by a Shigella spp. or an E.
coli. The subject may have or be at risk for chronic diarrhea or
chronic constipation.
[0018] The present invention also provides the use of a composition
described herein for the manufacture of a medicament, or for the
manufacture of a medicament for treating or ameliorating or
preventing a disease or a pathological or iatrogenic condition of
the colon.
[0019] Optionally the disease is a disease or condition
characterized by a dysfunctional or pathological composition of
colon microbiota, or the disease is a Clostridium difficile
colitis, or the disease or condition is a colitis, an autoimmune
colitis, an infectious colitis or an enterohemorrhagic colitis.
[0020] The term "and/or" means one or all of the listed elements or
a combination of any two or more of the listed elements.
[0021] The words "preferred" and "preferably" refer to embodiments
of the invention that may afford certain benefits, under certain
circumstances. However, other embodiments may also be preferred,
under the same or other circumstances. Furthermore, the recitation
of one or more preferred embodiments does not imply that other
embodiments are not useful, and is not intended to exclude other
embodiments from the scope of the invention.
[0022] The terms "comprises" and variations thereof do not have a
limiting meaning where these terms appear in the description and
claims.
[0023] Unless otherwise specified, "a," "an," "the," and "at least
one" are used interchangeably and mean one or more than one.
[0024] Also herein, the recitations of numerical ranges by
endpoints include all numbers subsumed within that range (e.g., 1
to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
[0025] For any method disclosed herein that includes discrete
steps, the steps may be conducted in any feasible order. And, as
appropriate, any combination of two or more steps may be conducted
simultaneously.
[0026] The above summary of the present invention is not intended
to describe each disclosed embodiment or every implementation of
the present invention. The description that follows more
particularly exemplifies illustrative embodiments. In several
places throughout the application, guidance is provided through
lists of examples, which examples can be used in various
combinations. In each instance, the recited list serves only as a
representative group and should not be interpreted as an exclusive
list.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 is a plot of microbial community taxonomic
distribution at the order level in a fecal transplant
bacteriotherapy recipient before and after receiving the transplant
in accordance with Example 1.
[0028] FIG. 2 is a plot of microbial community taxonomic
distribution at the family level in a fecal transplant
bacteriotherapy recipient before and after receiving the transplant
in accordance with Example 1.
[0029] FIG. 3 is a plot of community analysis done using the Yue
and Clayton's theta index showing that the post-transplantation
samples from a recipient clustering closely with each other and
with the donor's sample, compared to that of the recipient's
pre-transplantation sample.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0030] Before the present invention standard practices suggested
matching each recipient of fecal bacteriotherapy with a separate
donor, usually a close family member, or using the recipient's own
banked feces for later use. The rationale for these practices was
the idea that close family members have already shared their
pathogens, and that these kinds of gut microbiota would be somehow
better tolerated by the recipient's immune system because of
previous exposure. However, this resulted in duplicative screening,
burdening already debilitated patients with the task of finding a
suitable donor, pressure on the donor to provide the material and
potentially withholding important medical information, pressure to
decrease costs since costs were usually borne by the patient, time
delays associated with the screening, and pressure to accept donors
of suboptimal health status during donor selection. The
compositions presented herein result from a more standardized
manufacturing process with rigorous donor screening, multiple steps
of filtration that concentrate the microbiota and remove the bulk
of nonliving material, and optionally freeze/thaw it in a way that
preserves its viability. The compositions presented herein provide
a significant advantage by making useful compositions of colon
microflora readily available for use by a physician to treat a
patient. Moreover, it is much more aesthetically acceptable, as the
compositions are nearly odorless, are in concentrated form, and are
easily manipulated using standard laboratory practice.
[0031] The present invention provides compositions that include
fecal microbes. As used herein, the term "fecal microbes" refers to
microorganisms that are present in the gut, intestine, or colon,
preferably colon, of a normal healthy adult human. Such a
composition may be prepared by processing fecal material. As used
herein, the term "fecal material" refers to human stool.
Unprocessed fecal material contains non-living material and
biological material. The "non-living material" may include, but is
not limited to, dead bacteria, shed host cells, proteins,
carbohydrates, fats, minerals, mucus, bile, undigested fiber and
other foods, and other compounds resulting from food and metabolic
waste products and partial or complete digestion of food materials.
"Biological material" refers to the living material in fecal
material, and includes microbes including prokaryotic cells such as
bacteria and archea (e.g., living prokaryotic cells and spores that
can sporulate to become living prokaryotic cells), eukaryotic cells
such as protozoa and fungi, and viruses. In one embodiment,
"biological material" refers to the living material, e.g., the
microbes, eukaryotic cells, and viruses, that are present in the
colon of a normal healthy human.
[0032] Examples of prokaryotic cells that may be present in a
composition of the present invention include cells that are members
of the class Actinobacteria, such as the subclass Actinobacteridae
or Coriobacteridae, such as the order Bifidobacteriales or
Coriobacteriales, and/or such as the family Bifidobacteriaceae or
Coriobacteriaceae; members of the phylum Bacteroidetes, such as
class Bacteroidia, such as class Bacteroidales, and/or such as
family Bacteroidaceae or Rikenellaceae; members of the phylum
Firmicutes, such as class Bacilli, Clostridia, or Erysipelotrichi,
such as order Bacillales or Lactobacillales or Clostridales or
Erysipelotrichales, and/or such as family Paenibacillaceae or
Aeroccaceae or Lactobacillaceae or Streptococcaceae or
Catabacteriaceae or Peptococcaceae or Peptostreptococcaceae or
Ruminococcaceae or Clostridiaceae or Eubacteriaceae or
Lachnospiraceae or Erysipelotrichaceae; members of the phylum
Proteobacteria, such as class Alphaproteobacteria or
Betaproteobacteria or Gammaproteobacteria, such as order
Rhizobiales or Burkholderiales or Alteromonadales or
Enterobacteriales, and/or such as family Rhodobiaceae or
Burkholderiaceae or Shewanellaceae or Enterobacteriaceae; members
of the phylum Tenericutes, such as the class Mollicutes, such as
the order Entomoplasmatales, and/or such as the family
Spiroplasmataceae; and/or members of the class Verrucomicrobiae,
such as the order Verrucomicrobiales, and/or such as the family
Verrucomicrobiaceae.
[0033] In one embodiment a composition of the present invention may
include prokaryotic bacteria that are members of at least 1 phylum,
at least 2 phyla, at least 3 phyla, at least 4 phyla, at least 5
phyla, at least 6 phyla, at least 7 phyla, at least 8 phyla, at
least 9 phyla, or at least 10 phyla. In one embodiment a
composition of the present invention may include prokaryotic
bacteria that are members of at least 1 class, at least 2 classes,
at least 3 classes, at least 4 classes, at least 5 classes, at
least 6 classes, or at least 7 classes. In one embodiment a
composition of the present invention may include prokaryotic
bacteria that are members of at least 1 order, at least 2 orders,
at least 3 orders, at least 4 orders, at least 5 orders, at least 6
orders, or at least 7 orders. In one embodiment a composition of
the present invention may include prokaryotic bacteria that are
members of at least 1 family, at least 2 families, at least 3
families, at least 4 families, at least 5 families, at least 6
families, at least 7 families. In one embodiment a composition of
the present invention may include at least 5, at least 10, at least
20, or at least 30 different genera of prokaryotic bacteria. In one
embodiment a composition of the present invention may include at
least 10, at least 50, at least 100, at least 200, at least 300, or
at least 400 different species of prokaryotic bacteria.
[0034] In one embodiment a composition of the present invention
includes no greater than 5% weight of non-living material/weight
biological material (wt/wt), no greater than 2.5% (wt/wt), no
greater than 1% (wt/wt), no greater than 0.1% (wt/wt), no greater
than 0.01% (wt/wt), or no greater than 0.001% (wt/wt) nonliving
material. In one embodiment, the amount of non-living material in a
composition of the present invention is undetectable using
currently available techniques. For instance, living material can
be stained for biological activity, electron transport, DNA and RNA
for specific genes.
[0035] In one embodiment, the fecal material present in a
composition of the present invention does not include particles
(e.g., particles of non-living material and/or particles of
biological material) having a size of greater than 2.0 millimeters
(mm), greater than 1.0 mm, greater than 0.5 mm, greater than 0.25
mm, greater than 0 212 mm, greater than 0.180 mm, greater than
0.150 mm, greater than 0.125 mm, greater than 0.106 mm, greater
than 0.090 mm, greater than 0.075 mm, greater than 0.063 mm,
greater than 0.053 mm, greater than 0.045 mm, greater than 0.038
mm, greater than 0.032 mm, greater than 0.025 mm, greater than
0.020 mm, greater than 0.01 mm, or greater than 0.2 mm. Non-fecal
material present in a composition may include particles having a
size of greater than 2.0 mm, greater than 1.0 nun, greater than 0.5
mm, greater than 0.25 mm, greater than 0.212 mm, greater than 0.180
mm, greater than 0.150 mm, greater than 0.125 mm, greater than
0.106 mm, greater than 0.090 mm, greater than 0.075 mm, greater
than 0.063 mm, greater than 0.053 mm, greater than 0.045 mm,
greater than 0.038 mm, greater than 0.032 mm, greater than 0.025
mm, greater than 0.020 mm, greater than 0.01 mm, or greater than
0.2 mm. In one embodiment, the fecal material present in a
composition of the present invention consists of, or consists
essentially of, particles of non-living material and/or biological
material having a size that will pass through a sieve having a
sieve size of 2.0 nun, 1.0 mm, 0.5 mm, 0.25 mm, 0.212 mm, 0.180 mm,
0.150 mm, 0.125 mm, 0.106 mm, 0.090 mm, 0.075 mm, 0.063 mm, 0.053
mm, 0.045 mm, 0.038 mm, 0.032 mm, 0.025 mm, 0.020 mm, 0.01 mm, or
0.2 mm. Thus, in such an embodiment, the fecal material present in
a composition has a size that is less than or equal to 2.0 mm, less
than or equal to 1.0 mm, less than or equal to 0.5 mm, less than or
equal to 0.25 mm, less than or equal to 0.212 mm, less than or
equal to 0.180 mm, less than or equal to 0.150 mm, less than or
equal to 0.125 mm, less than or equal to 0.106 mm, less than or
equal to 0.090 mm, less than or equal to 0.075 mm, less than or
equal to 0.063 mm, less than or equal to 0.053 mm, less than or
equal to 0.045 mm, less than or equal to 0.038 mm, less than or
equal to 0.032 mm, less than or equal to 0.025 mm, less than or
equal to 0.020 mm, less than or equal to 0.01 mm, or less than or
equal to 0.2 mm. The sieve size may be based on the US Standard
sieve sizes of, for instance, 10, 18, 35, 60, 70, 80, 100, 120,
140, 170, 200, 230, 270, 325, or 400.
[0036] A composition of the present invention may optionally
include a cryoprotectant. A cryoprotectant is a compound that
maintains the viability of fecal microbes when frozen.
Cryoprotectants are known in the art and used routinely to protect
microbes when exposed to freezing conditions. Examples include, but
are not limited to, amino acids such as alanine, glycine, proline;
simple sugars such as sucrose, glucose, lactose, ribose, and
trehalose; and other compounds such as dimethyl sulfoxide (DMSO),
and glycerol. The amount of cryoprotectant present in a composition
described herein may vary depending on the cryoprotectant used and
the temperature to be used for freezing (e.g., -20.degree. C.,
-80.degree. C., or a different temperature). The amount of
cryoprotectant that can be used is known to the skilled person or
may be easily determined using routine experimentation. In one
embodiment, a composition of the present invention may include
glycerol at a concentration of 10%.
[0037] In one embodiment a composition of the present invention
does not include pathogenic biological material. In one embodiment,
fecal material is from a person that has undergone a medical
history, a physical examination, and laboratory testing. The
evaluation of medical history may include, but is not limited to,
risk of infectious agents, presence of gastrointestinal
co-morbidities, factors that can or do affect the composition of
the intestinal microbiota, and systemic medical conditions.
Exclusion criteria regarding risk of infectious agents may include,
but are not limited to, known viral infection with Hepatitis B, C
or HIV; known exposure to HIV or viral hepatitis at any time; high
risk behaviors including sex for drugs or money, men who have sex
with men, more than one sexual partner in the preceding 12 months,
any past use of intravenous drugs or intranasal cocaine, history of
incarceration; tattoo or body piercing within 12 months; travel to
areas of the world where risk of traveler's diarrhea is higher than
the US; and current communicable disease, e.g., upper respiratory
viral infection.
[0038] Exclusion criteria regarding gastrointestinal co-morbidities
include, but are not limited to, history of irritable bowel
syndrome, wherein specific symptoms may include frequent abdominal
cramps, excessive gas, bloating, abdominal distension, fecal
urgency, diarrhea, constipation; history of inflammatory bowel
disease such as Crohn's disease, ulcerative colitis, microscopic
colitis; chronic diarrhea; chronic constipation or use of
laxatives; history of gastrointestinal malignancy or known colon
polyposis; history of any abdominal surgery, e.g., gastric bypass,
intestinal resection, appendectomy, cholecystectomy, and the like;
use of probiotics or any other over the counter aids used by the
potential donor for purpose of regulating digestion, but yogurt and
kefir products may be allowed if taken merely as food rather than
nutritional supplements.
[0039] Exclusion criteria regarding factors that can or do affect
the composition of the intestinal microbiota include, but are not
limited to, antibiotics for any indication within the preceding 6
months; any prescribed immunosuppressive or anti-neoplastic
medications.
[0040] Exclusion criteria regarding systemic medical conditions
include, but are not limited to, established or emerging metabolic
syndrome, where criteria used for definition here are stricter than
established criteria, including history of increased blood
pressure, history of diabetes or glucose intolerance; known
systemic autoimmunity, e.g., connective tissue disease, multiple
sclerosis; known atopic diseases including asthma or eczema;
chronic pain syndromes including fibromyalgia, chronic fatigue
syndrome; ongoing (even if intermittent) use of any prescribed
medications, including inhalers or topical creams and ointments;
neurologic, neurodevelopmental, and neurodegenerative disorders
including autism, Parkinson's disease.
[0041] Exclusion criteria on physical examination may include, but
are not limited to, general, such as body mass index >26
kg/m.sup.2, central obesity defined by waste:hip ratio >0.85
(male) and >0.80 (female); blood pressure >135 mmHg systolic
and >85 mmHg diastolic; skin--presence of a rash, tattoos or
body piercing placed within a year, jaundice; enlarged lymph nodes;
wheezing on auscultation; hepatomegaly or stigmata of liver
disease; swollen or tender joints; muscle weakness; abnormal
neurologic examination.
[0042] Exclusion criteria on laboratory testing may include, but is
not limited to, positive stool Clostridium difficile toxin B tested
by PCR; positive stool cultures for any of the routine pathogens
including Salmonella, Shigella, Yersinia, Campylobacter, E. coli
0157:H7; abnormal ova and parasites examination; positive Giardia,
Cryptosporidium, or Helicobacter pylori antigens; positive
screening for any viral illnesses, including HIV 1 and 2, Viral
Hepatitis A IgM, Hepatitis surface antigen and core Ab; abnormal
RPR (screen for syphilis); any abnormal liver function tests
including alkaline phosphatase, aspartate aminotransaminase,
alanine aminotransferase; raised serum triglycerides >150 mg/dL;
HDL cholesterol <40 mg/dL (males) and <50 mg/dL (females);
high sensitivity CRP >2.4 mg/L; raised fasting plasma glucose
(>100 mg/dL).
[0043] The compositions of the present invention may be included in
a diversity of pharmaceutically acceptable formulations. In one
embodiment, a formulation may be a fluid composition. Fluid
compositions include, but are not limited to, solutions,
suspensions, dispersions, and the like. In one embodiment, a
formulation may be a solid composition. Solid compositions include,
but are not limited to, powder, granule, compressed tablet, pill,
capsule, chewing gum, wafer, and the like. Those formulations may
include a pharmaceutically acceptable carrier to render the
composition appropriate for administration to a subject. As used
herein "pharmaceutically acceptable carrier" includes
pharmacologically inactive compounds compatible with pharmaceutical
administration. The compositions of the present invention may be
formulated to be compatible with its intended route of
administration. A composition of the present invention may be
administered by any method suitable for depositing in the
gastrointestinal tract, preferably the colon, of a subject.
Examples of routes of administration include rectal administration
(e.g., by suppository, enema, upper endoscopy, upper push
enteroscopy, or colonoscopy), intubation through the nose or the
mouth (e.g., by nasogastric tube, nasoenteric tube, or nasal
jejunal tube), or oral administration (e.g., by a solid such as a
pill, tablet, or capsule, or by liquid).
[0044] For therapeutic use in the method of the present invention,
a composition may be conveniently administered in a form containing
one or more pharmaceutically acceptable carriers. Suitable carriers
are well known in the art and vary with the desired form and mode
of administration of the composition. For example, they may include
diluents or excipients such as fillers, binders, wetting agents,
disintegrators, surface-active agents, glidants, lubricants, and
the like. Typically, the carrier may be a solid (including powder),
liquid, or combinations thereof. Each carrier is preferably
"acceptable" in the sense of being compatible with the other
ingredients in the composition and not injurious to the subject.
The carrier is preferably biologically acceptable and inert, i.e.,
it permits the composition to maintain viability of the biological
material until delivered to the appropriate site.
[0045] Oral compositions may include an inert diluent or an edible
carrier. For the purpose of oral therapeutic administration, the
active compound can be incorporated with excipients and used in the
form of tablets, troches, or capsules, e.g., gelatin capsules. Oral
compositions can also be prepared by combining a composition of the
present invention with a food. In one embodiment a food used for
administration is chilled, for instance, ice cream.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0046] The active compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0047] The active compounds may be prepared with carriers that will
protect the compound against rapid elimination from the body, such
as a controlled release formulation, including implants.
Biodegradable, biocompatible polymers can be used, such as ethylene
vinyl acetate, polyanhydrides, polyglycolic acid, collagen,
polyorthoesters, and polylactic acid. Such formulations can be
prepared using standard techniques. The materials can also be
obtained commercially from, for instance, Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions can also be used as
pharmaceutically acceptable carriers. These can be prepared
according to methods known to those skilled in the art.
[0048] In one embodiment, a composition may be encapsulated. For
instance, when the composition is to be administered orally, the
dosage form is formulated so the composition is not exposed to
conditions prevalent in the gastrointestinal tract before the
colon, e.g., high acidity and digestive enzymes present in the
stomach and/or intestine. The encapsulation of compositions for
therapeutic use is routine in the art. Encapsulation may include
hard-shelled capsules, which may be used for dry, powdered
ingredients soft-shelled capsules. Capsules may be made from
aqueous solutions of gelling agents such as animal protein (e.g.,
gelatin), plant polysaccharides or derivatives like carrageenans
and modified forms of starch and cellulose. Other ingredients may
be added to a gelling agent solution such as plasticizers (e.g.,
glycerin and or sorbitol), coloring agents, preservatives,
disintegrants, lubricants and surface treatment.
[0049] A composition may be prepared by obtaining a fecal sample
from an appropriate donor and blending with a diluent. Useful
diluents include aqueous solutions that are routinely used for
manipulating microbes, eukaryotic cells, and/or viruses. Useful
diluents may include constituents to maintain physiological buffer,
osmolarity, and the like. The diluent is preferably sterile and/or
non-allergenic. An example of a diluent includes, but is not
limited to, phosphate buffered saline at pH 7. In one embodiment, 1
part donor feces may be combined with 5 parts diluent (e.g., 50
grams of donor feces may be combined with 250 mls diluent) and
blended. In one embodiment, the oxygen in the blending chamber may
be decreased or removed by purging with an inert gas such as
nitrogen or argon prior to blending. Such anaerobic conditions may
be useful to maintain viability of most anaerobic bacteria present
in a colon. The sample may be blended multiple times and/or more
diluent may be added until a consistency is achieved that will
permit the following steps to occur. In one embodiment, anaerobic
conditions are not used in steps following the blending. It was
found that anaerobic conditions were not necessary in the steps
following the blending, and this was unexpected and surprising
since a substantial percentage of prokaryotic cells in fecal
material are strict anaerobes, and exposure to oxygen kills them.
After the blending, the solutions used for washing and resuspension
did not need to be purged of oxygen, and manipulation of the
microbiota in an oxygen-free cabinet or glove box was not
needed.
[0050] Not all microbes and eukaryotic cells present in an
individual's colon can be cultured, thus, in one embodiment
conditions for preparing a composition include the use of
temperatures that decrease the replication of the microbes and
eukaryotic cells. In one embodiment, the conditions used for
preparation are maintained below 37.degree. C. For instance, the
conditions used for preparation are maintained at a temperature of
no greater than 30.degree. C., no greater than 20.degree. C., no
greater than 10.degree. C., or no greater than 5.degree. C. In one
embodiment, conditions are used such that replication of the
microbes and eukaryotic cells does not occur. When the conditions
used to prepare a composition of the present invention include
lower temperatures to minimize replication and cell death, the
biological material present in a composition includes a population
of microbes, eukaryotic cells, and viruses that is essentially
identical to a population of microbes, eukaryotic cells, and
viruses present in the colon or feces of a normal healthy human,
e.g., the donor from whom the fecal sample was obtained.
[0051] Removal of non-living material may be achieved by passing
the blended sample through a sieve with a sieve size of no greater
than 2.0 mm, no greater than 1.0 mm, no greater than 0.5 min, no
greater than 0.25 mm, no greater than 0.212 mm, no greater than
0.180 mm, no greater than 0.150 mm, no greater than 0.125 mm, no
greater than 0.106 mm, no greater than 0.090 mm, no greater than
0.075 mm, no greater than 0.063 mm, no greater than 0.053 mm, no
greater than 0.045 mm, no greater than 0.038 mm, no greater than
0.032 mm, no greater than 0.025 mm, no greater than 0.020 nun, no
greater than 0.01 mm, or no greater than 0.2 mm. In one embodiment,
the blended sample is prepared by passing it through a sieve with a
sieve size of 0.25 mm and collecting the filtrate. In one
embodiment, the blended sample is passed through sieves with
progressively smaller sieve sizes until final passage through a
sieve size of 0.25 mm. For instance, if a total of four sieves are
used the sieve size of the first sieve may be 2 mm, followed by 1
mm, followed by 0.5 mm, and then followed by 0.25 mm. The final
filtrate may be collected in a centrifuge tube, and centrifuged at
a speed sufficient to pellet the biological material, for instance,
10,000.times.g for 10 minutes at 4.degree. C. The supernatant is
removed, the cells are resuspended in diluent, optionally
centrifuged again, for instance at 10,000.times.g for 10 minutes at
4.degree. C. The final supernatant is discarded and the cells are
resuspended in an aqueous solution (e.g., diluent, cryoprotectant,
and the like, or a combination thereof). In one embodiment, the
volume of the blended mixture is decreased through the steps of
sieving and washing. For instance, in one embodiment, the volume is
decreased to 14% of the volume used in the blending (e.g., from 250
mls to 35 mls). In one embodiment, the volume of the blended
mixture is decreased through the steps of sieving and washing to
result in between 1.times.10.sup.10 and 5.times.10.sup.10 cells in
a volume that is subsequently administered to a subject. This
process results in an extract of feces that is highly enriched for
all colon microbiota that are able to pass through a sieve as
described above, and can be centrifuged at 10,000.times.g for 10
minutes. As used herein, "enriched" refers to increasing the
abundance of biological material relative to non-living material,
such that biological material constitutes a significantly higher
proportion compared to the fecal material before the enrichment.
The term "enriched" refers to those situations in which a person
has intervened to elevate the proportion of biological
material.
[0052] The amount of aqueous solution added may be in an amount to
result in a single dosage having an appropriate number of cells. In
one embodiment, a single dosage may include between
1.times.10.sup.10 and 5.times.10.sup.10 cells, for instance,
3.times.10.sup.10 cells. Since most biological material is
difficult or impossible to culture, a hemocytometer may be used to
determine the number of cells.
[0053] In one embodiment the resulting pellet may be suspended in
half the original volume of diluent containing 10% glycerol. The
sample may be used immediately, or may be frozen, for instance, at
-80.degree. C., for later use. When freezing, the sample may be
left in a centrifuge tube, or may be in a different container. In
one embodiment, the container is one that increases the surface
area of the sample. For instance, the sample may be placed in an IV
bag. When the frozen sample is to be used, it may be thawed on ice
and then transplanted into the recipient. It was found that
freezing the compositions described herein did not result in
destruction of its curative potential. In one embodiment the sample
resulting from centrifugation may be processed for long term
storage of 1 year or longer. The ability to store such a sample
provides a level of flexibility that was not possible with other
methods. For instance, it was necessary to quickly identify a
donor, rapidly process a fecal sample from the donor, and use it
immediately. Examples of useful processing methods include, but are
not limited to, freezing, freeze drying, spray drying,
lyophilization, vacuum drying, air drying, or other forms of
evaporative drying. Processing of a composition of the present
invention may include the production of a powder following any
drying procedure.
[0054] The use of sieves to extract biological material from fecal
material unexpectedly resulted in a composition which was nearly
odorless. This was not expected because feces normally have a
distinctive odor and this was surprising to be removed by the
minimal manipulation used. This is a significant advantage as it
takes a method that is unaesthetic and so distasteful that some
patients and staff refuse to take part, and changes it into a
method that is easily practiced in a normal clinical setting or at
home. As used herein, "odorless" means there is a decreased amount
of volatile organic molecules present, and the decreased amount of
volatile organic molecules present can be easily detected by a
person comparing the material before processing with the material
after processing.
[0055] The present invention is further directed to methods of
using the compositions described herein. A method of the present
invention includes administering to a subject in need thereof an
effective amount of a composition described herein. The
administering is under conditions suitable for deposition of the
composition in a region of the large or small intestine such that
the biological material in the composition colonizes the colon. For
instance, administration may be into upper gastrointestinal tract,
as well as lower gastrointestinal tract, e.g., the terminal ileum,
cecum, colonic areas containing diverticulosis, and rectum. In one
embodiment the administering may be oral, such as by tablet. In one
embodiment the administering may be by intubation, such as by
nasogastric tube. In one embodiment the administering may be
rectal, for instance by a colonoscope, enema, or suppository.
Conditions that are "suitable" for an event to occur, or "suitable"
conditions are conditions that do not prevent such events from
occurring. Thus, these conditions permit, enhance, facilitate,
and/or are conducive to the event. As used herein, an "effective
amount" relates to a sufficient amount of a composition described
herein, to provide the desired effect. For instance, in one
embodiment an "effective amount" is an amount effective to
alleviate one or more symptoms and/or signs of the disease as
described herein. In some embodiments, an effective amount is an
amount that is sufficient to effect a reduction in a symptom and/or
sign associated with a disease, such as diarrhea or C. difficile. A
reduction in a symptom and/or a sign is, for instance, at least
10%, at least 20%, at least 30%, at least 40%, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, or at least
100% in a measured sign as compared to a control, a non-treated
subject, or the subject prior to administration of the composition.
In one embodiment, an effective amount is an amount sufficient to
result in at least 1.times.10.sup.10, at least 3.times.10.sup.10,
or at least 5.times.10.sup.10 cells delivered to the colon. It will
be understood, however, that the total dosage of the compositions
as disclosed herein will be decided by the attending physician
within the scope of sound medical judgment. The exact amount
required will vary depending on factors such as the type and extent
of disease being treated.
[0056] In one embodiment, a method of the present invention
includes treating certain diseases in a subject in need of
treatment. The subject may be a mammal, such as a human. In some
embodiments animal models may be used, such as a mammal, including
a rat, a mouse, a hamster, a gerbil, or a primate. As used herein,
the term "disease" refers to any deviation from or interruption of
the normal structure or function of a part, organ, or system, or
combination thereof, of a subject that is manifested by a
characteristic symptom or clinical sign. Diseases include those
characterized by dysfunctional composition of colon microbiota.
Such diseases include, but are not limited to, colitis, including
autoimmune colitis (e g, inflammatory bowel disease, ulcerative
colitis, Crohn's disease, irritable bowel syndrome) and infectious
colitis. Examples of infectious colitis include, but are not
limited to Clostridium difficile colitis (e.g., acute C. difficile
colitis, relapsing C. difficile colitis, or severe C. difficile
colitis) and enterohemorrhagic colitis (e.g., a colitis caused by
Shigella spp. or E. coli). Other examples of diseases include, but
are not limited to, chronic diarrhea; chronic constipation,
metabolic syndrome and obesity, atopic diseases including asthma,
eczema, eosinophilic disorders of the GI tract, systemic
autoimmunity including rheumatoid arthritis, systemic lupus
erythematosis, multiple sclerosis, etc., chronic pain disorders
such fibromyalgia, chronic fatigue syndrome, neurodegenerative
disorders, eating disorders, and malnutrition.
[0057] As used herein, the term "symptom" refers to subjective
evidence of disease or condition experienced by the patient and
caused by disease. As used herein, the term "clinical sign," or
simply "sign," refers to objective evidence of a disease present in
a subject. Symptoms and/or signs associated with diseases referred
to herein and the evaluation of such signs are routine and known in
the art. Typically, whether a subject has a disease, and whether a
subject is responding to treatment, may be determined by evaluation
of signs associated with the disease.
[0058] Treatment of a disease can be prophylactic or,
alternatively, can be initiated after the development of a disease.
Treatment that is prophylactic, for instance, initiated before a
subject manifests signs of a disease, is referred to herein as
treatment of a subject that is "at risk" of developing a disease.
An example of a subject that is at risk of developing a disease is
a person having a risk factor. An example of a risk factor for
Clostridium difficile colitis is antibiotic therapy of the
gastrointestinal tract. Treatment can be performed before, during,
or after the occurrence of the diseases described herein. Treatment
initiated after the development of a disease may result in
decreasing the severity of the signs of the disease, or completely
removing the signs.
[0059] In one embodiment, a method of the present invention
includes transplanting a microbiota from a donor to a
recipient.
[0060] In one embodiment, a method of the present invention
includes increasing the relative abundance of members of the phylum
Firmicutes, such as a nonpathogenic member of the class Clostridia,
and/or members of the phylum Bacteroidetes, in a recipient's colon.
The phrase "relative abundance" refers to number of members of a
phylum or class compared to the number of members of all other taxa
in a recipient's colon. Such a comparison can be expressed as a
percent. In one embodiment, the relative abundance of
non-pathogenic members of the class Clostridia in a recipient's
colon after the administration may be increased by at least 5%, at
least 10%, at least 20%, or at least 50%, compared to the
recipient's colon before the administration. In one embodiment, the
relative abundance of members of the phylum Firmicutes in a
recipient's colon after the administration may be increased by at
least 5%, at least 10%, at least 20%, or at least 50% compared to
the recipient's colon before the administration. The change in the
abundance may be determined at, for instance, 3 days, 10 days, 15
days, or 25 days after the administration of fecal microbiota.
[0061] In one embodiment, a method of the present invention
includes decreasing the relative abundance of members of the phylum
Proteobacteria in a recipient's colon. In one embodiment, the
relative abundance of members of the phylum Proteobacteria in a
recipient's colon after the administration may be decreased by at
least 10%, at least 20%, at least 30%, or at least 40% compared to
the recipient's colon before the administration. The change in the
abundance of members of the phylum Proteobacteria may be determined
at, for instance, 3 days, 10 days, 15 days, or 25 days after the
administration.
[0062] In one embodiment, the existing microbiota does not need to
be cleared prior to administration of a composition of the present
invention. In other embodiments clearance of the microbiota may be
necessary. Methods for clearance of existing microbiota are known
and routine. In one example, clearance can be accomplished by
administering a cocktail of antibiotics for one week until a day
prior to transplant. An example of a useful cocktail is
Metronidazole (1000 mg twice daily), Rifaximin (550 mg twice
daily), Vancomycin (500 mg twice daily), and Neomycin (1000 mg
twice daily).
[0063] The present invention is illustrated by the following
examples. It is to be understood that the particular examples,
materials, amounts, and procedures are to be interpreted broadly in
accordance with the scope and spirit of the invention as set forth
herein.
Example 1
[0064] Clostridium difficile associated disease is a major known
complication of antibiotic therapy. The pathogen is normally held
in check by native colon microbiota, but this level of protection
may be lost when these microbial communities are suppressed by
antibiotics. Antibiotics used to treat C. difficile infection may
also perpetuate its recurrence by continued suppression of normal
microbiota. Thus, a significant fraction of patients suffer from
recalcitrant C. difficile infection, and recalcitrant C. difficile
infection is associated with significant morbidity. Fecal
bacteriotherapy is an increasingly used method used to break the
cycle of C. difficile infection recurrence presumably through
restoration of normal intestinal microbial communities. We
previously reported, in one clinical case, that bacteriotherapy of
colon microbiota resulted in the replacement of a host's microbiota
by that of the donor (Khoruts, et al., 2010, J. Clin.
Gastroenterol., 44(5):354). In order to obtain a greater
understanding of the composition and stability of microbial
communities before and after bacteriotherapy, we have analyzed
amplified 16S rRNA regions of fecal DNA (V5 and V6) by using a
pyrosequencing technology (an Illumina HiSeq2000 or other Illumina
platforms). Additional individuals are currently being processed
and analyzed.
[0065] Introduction [0066] Clostridium difficile is an emerging
pathogen and the most common cause of nosocomial diarrhea. [0067]
Infections are often associated with antibiotic therapy, where the
protective effect provided by the normal intestinal flora is
disrupted. [0068] C. difficile infection is often controlled by
additional antimicrobial therapy, but approximately 20% of patients
develop refractory disease resulting in recurrent diarrhea. [0069]
Bacteriotherapy, in the form of a fecal transplantation, has been
shown to successfully treat refractory C. difficile infection.
[0070] Next generation sequencing technologies have allowed for a
deeper interrogation of the intestinal microflora and was used in
our study to examine changes in microbial community structure after
transplantation.
[0071] Donor fecal material was obtained from the patient's son,
who was tested for infectious disease, including C. difficile,
Hepatitis A, B, or C viruses, HIV virus, Salmonella, Campylobacter,
Yersinia, Shigella, E. Coli 0157:H7, Helicobacter pylori, Treponema
pallidum, Giardia, and Cryptosporidium.
[0072] The patient was infused with donor fecal material by
colonoscopy, which revealed severe, extensive diverticulosis in the
sigmoid colon. The donor's fecal material was deposited into the
cecum. Symptoms consistent with C. difficile infection were
resolved within days of bacteriotherapy.
[0073] Methods [0074] Patient fecal samples were collected at day
-31 before the fecal transplant bacteriotherapy and at days 5, 21,
46, 95, 132, 159, 188, and 227 post transplantation. A donor fecal
sample was collected the day of the procedure and deposited into
the recipient's cecum. [0075] DNA was extracted from fecal
materials using a MOBIO ultra-clean fecal DNA kit (MOBIO
Laboratories, Inc., Carlsbad, Calif.) as directed by the
manufacturer. Triplicate samples were extracted and pooled. [0076]
The V6 hypervariable region of the bacteria 16S rRNA gene was
amplified using 50 ng of extracted DNA as template. Barcoded
primers were used for multiplex sequencing (Kysela et al., 2005,
Environmental Microbiology 7:356-64, and Claesson et al., Nucleic
Acids Research, 2010, Vol. 38, No. 22 e200 doi:10.1093/narigkq873).
Triplicate samples were prepared and pooled. [0077] Amplified
samples were mixed in equimolar ratios and sequenced at the
National Center for Genomic Research (NCGR) using the Illumina
sequencing platform. [0078] Sequence data was analyzed using MOTHUR
and the SILVA reference database (Scholss, 2009, Appl. Environ.
Microbiol., 75(23):7537-7541. The taxonomy of operational taxonomic
units (OTUs) were assigned at the 97% similarity using the
GreenGenes reference files. (FIGS. 1 and 2) [0079] Principal
component analysis was done using Yue and Clayton's Theta
calculation (Yue and Clayton, 2005, Commun. Stat. Theor. Methods,
34:2123-2131). Accumulation curves were calculated based on 97% OTU
similarities. (FIG. 3)
[0080] Results & Discussion [0081] Greater than 40% of the
sequences obtained from the recipient's pretransplantation sample
(day -31) belonged to unclassified Mollicutes strains or the
Gammaproteobacteria. [0082] In contrast, the donor's and
recipient's post-transplantation samples were dominated by
Firmicutes. Unclassified members of the Clostridiales and the
Ruminococcaceae family were abundant. [0083] Community analysis
done using the Yue and Clayton's theta index showed that the
post-transplantation samples clustered more closely with each other
and with the donor sample, compared to that of the recipient's
pre-transplantation sample. [0084] Sequence analysis indicated that
the taxa present in the recipient's pre- and post-transplant fecal
samples differed considerably, suggesting that fecal
bacteriotherapy was successful in altering the patient's intestinal
microflora. [0085] The transplanted microbial community in the
recipient's intestine remained fairly stable after 7.5 months post
transplantation.
[0086] Surprisingly, sequences representing the Bacteroidales were
in fairly low abundance in all of the samples analyzed.
TABLE-US-00001 TABLE 1 Pyrosequencing Metrics Sample Day Seqs OTUs
(97%) Coverage Shannon Index -31 1335704 15600 0.992 3.844 Donor
2892413 34945 0.992 4.663 +5 2631872 28948 0.992 4.004 +21 2909055
30038 0.993 4.047 +46 3604923 35864 0.993 4.277 +95 2216996 27782
0.991 4.631 +132 2927800 26510 0.994 3.746 +159 2691936 30990 0.992
4.551 +188 1649565 18417 0.993 3.781 +277 3073102 31162 0.993
3.852
Example 2
[0087] Donor Screening for Fecal Microbiota Material
Preparation
[0088] The donor undergoes a complete medical history and physical
examination. In addition, a full-length donor history questionnaire
is completed as recommended by the FDA for blood donors, and
potential donors saying yes to any of the questions are excluded
(http://wwvv.fda.gov/downloads/BiologicsBloodVaccines/BloodBloodProducts/-
ApprovedPr
oducts/LicensedProductsBLAs/BloodDonorScreening/UCM213552.pdf).
However, as gut microbiota have been associated or postulated to be
involved with multiple medical conditions, the process of selection
is more rigorous than that of the blood donors and includes
virtually any systemic illness.
[0089] Inclusion Criteria [0090] 1. Age >18 [0091] 2. Ability to
provide informed consent.
[0092] Exclusion Criteria [0093] I. Medical History [0094] A. Risk
of infectious agent. [0095] 1. Known viral infection with Hepatitis
B, C or HIV [0096] 2. Known exposure to HIV or viral hepatitis at
any time [0097] 3. High risk behaviors including sex for drugs or
money, men who have sex with men, more than one sexual partner in
the preceding 12 months, any past use of intravenous drugs or
intranasal cocaine, history of incarceration. [0098] 4. Tattoo or
body piercing within 12 months. [0099] 5. Travel to areas of the
world where risk of traveler's diarrhea is higher than the US.
[0100] 6. Current communicable disease, e.g., upper respiratory
viral infection. [0101] B. Gastrointestinal comorbidities. [0102]
1. History of irritable bowel syndrome. Specific symptoms may
include frequent abdominal cramps, excessive gas, bloating,
abdominal distension, fecal urgency, diarrhea, constipation. [0103]
2. History of inflammatory bowel disease such as Crohn's disease,
ulcerative colitis, microscopic colitis. [0104] 3. Chronic
diarrhea. [0105] 4. Chronic constipation or use of laxatives.
[0106] 5. History of gastrointestinal malignancy or known colon
polyposis. [0107] 6. History of any abdominal surgery, e.g.,
gastric bypass, intestinal resection, appendectomy,
cholecystectomy, etc. [0108] 7. Use of Probiotics or any other over
the counter aids used by the potential donor for purpose of
regulating digestion. Yogurt and kefir products are allowed if
taken merely as food rather than nutritional supplements. [0109] C.
Factors that can or do affect the composition of the intestinal
microbiota. [0110] 1. Antibiotics for any indication within the
preceding 6 months. [0111] 2. Any prescribed immunosuppressive or
anti-neoplastic medications. [0112] D. Systemic Medical Conditions.
[0113] 1. Metabolic Syndrome, established or emerging. Criteria
used for definition here are stricter than any established
criteria. These include history of increased blood pressure,
history of diabetes or glucose intolerance. [0114] 2. Known
systemic autoimmunity, e.g., connective tissue disease, multiple
sclerosis. [0115] 3. Known atopic diseases including asthma or
eczema. [0116] 4. Chronic pain syndromes including fibromyalgia,
chronic fatigue syndrome. [0117] 5. Ongoing (even if intermittent)
use of any prescribed medications, including inhalers or topical
creams and ointments. [0118] 6. Neurologic, neurodevelopmental, and
neurodegenerative disorders including autism, Parkinson's disease.
[0119] II. Exclusion Criteria on Physical Examination. [0120] 1.
General. Body mass index >26 kg/m.sup.2, central obesity defined
by waste:hip ratio >0.85 (male) and >0.80 (female). [0121] 2.
Blood pressure >135 mmHg systolic and >85 mmHg diastolic.
[0122] 3. Skin--presence of a rash, tattoos or body piercing placed
within a year, jaundice. [0123] 4. Enlarged lymph nodes. [0124] 5.
Wheezing on auscultation. [0125] 6. Hepatomegaly or stigmata of
liver disease. [0126] 7. Swollen or tender joints. Muscle weakness.
[0127] 8. Abnormal neurologic examination. [0128] III. Exclusion
Criteria on Laboratory Testing. [0129] 1. Positive stool
Clostridium difficile toxin B tested by PCR. [0130] 2. Positive
stool cultures for any of the routine pathogens including
Salmonella, Shigella, Yersinia, Campylobacter, E. coli 0157:H7.
[0131] 3. Abnormal ova and parasites examination. [0132] 4.
Positive Giardia, Cryptosporidium, or Helicobacter pylori antigens.
[0133] 5. Positive screening for any viral illnesses, including HIV
1 and 2, Viral Hepatitis A IgM, Hepatitis surface antigen and core
Ab. [0134] 6. Abnormal RPR (screen for syphilis). [0135] 7. Any
abnormal liver function tests including alkaline phosphatase,
aspartate aminotransaminase, alanine aminotransferase. [0136] 8.
Raised serum triglycerides >150 mg/dL [0137] 9. BDL cholesterol
<40 mg/dL (males) and <50 mg/dL (females) [0138] 10. High
sensitivity CRP >2.4 mg/L [0139] 11. Raised fasting plasma
glucose (>100 mg/dL)
Example 3
[0140] Fecal Sample Processing Donor fecal material is immediately
chilled on ice for transport to the laboratory.
[0141] Samples are processed within one hour after collection.
[0142] Fecal samples are homogenized by mixing 50 g of donor feces
and 250 ml of sterile phosphate buffered saline, pH 7, (PBS) in a
Waring Blender. The blending chamber is purged with nitrogen gas
for several minutes to remove oxygen prior to homogenization.
Samples are blended three times on the lowest setting for 20
seconds. Additional PBS or blending cycles may be added depending
on the consistency of the fecal suspension. Blended samples are
passed through a series of four sieves with pore sizes of 2.0 mm,
1.0 mm, 0.5 mm and 0.25 mm (W.S. Tyler Industrial Group, Mentor,
Ohio). The sieves were based on US standard sieve sizes of 10, 18,
35, and 60 for 2.0 mm, 1.0 mm, 0.5 mm and 0.25 mm, respectively.
The final filtrate passing through the sieves (less than 0.25 mm
fraction) is collected in 50 ml conical centrifuge tubes and
centrifuged at 4,000 rpm (about 4,000.times.g) for 10 minutes at
4.degree. C. The supernatant is discarded and the pellet is
suspended in one half the original volume of PBS (e.g. 125 ml)
containing 10% glycerol. The samples are used immediately, or
stored frozen at -80.degree. C. and thawed on ice before
transplantation.
Example 4
[0143] This example reports clinical experience with 43 consecutive
patients that were treated for recurrent CDI C. difficile infection
(CDI). During this time donor identification and screening was
simplified by moving from patient-identified individual donors to
standard volunteer donors. Material preparation shifted from the
endoscopy suite to a standardized process in the laboratory, and
ultimately to banking frozen processed fecal material that is ready
to use when needed.
[0144] Standardization of material preparation significantly
simplified the practical aspects of treatment without loss of
apparent efficacy in clearing recurrent CDI. Approximately 30% of
the patients had underlying inflammatory bowel disease, and treat
went was equally effective in this group. Several key steps in
standardization of donor material preparation significantly
simplified the clinical practice of treatment of recurrent CDI in
patients failing antibiotic therapy. This is also reported in
Hamilton et al., Am. J. Gastroenterol., 2012,
doi:10.1038/ajg.2011.482.
[0145] Methods
[0146] Patients
[0147] This report includes the first 43 patients who received
fecal microbiota transplantation (FMT) for recurrent CDI. All
patients were identified by direct referral from clinicians at
infectious disease and gastroenterology practices in the
Minneapolis and St. Paul metropolitan area. Inclusion criteria for
FMT included a history of symptomatic, toxin-positive, infection by
C. difficile and at least two documented subsequent recurrences
despite use of standard antibiotic therapy. At least one failed
antibiotic regimen had to include a minimum of a 6 week course of
tapered or pulsed vancomycin dosage, or at least a one month
vancomycin course followed by a minimum two week rifaximin
"chaser." The only exclusion criteria in the protocol were age
<18 and medical fragility from non-C. difficile problems
resulting in life expectancy of <1 year. In the latter situation
we advised patients that the best therapeutic option was an
indefinite course of vancomycin. All patients gave informed consent
for FMT via colonoscopy, recognizing relatively limited experience
with this treatment approach and the intrinsic unknowns associated
with its use. The Institutional Review Board at the University of
Minnesota approved prospective collection of clinical outcome data,
while recognizing this experience does not constitute a clinical
trial, and as such was not designed to test the efficacy of FMT in
comparison with any other therapeutic options.
[0148] Donor Identification and Screening
[0149] At the start of the program patients were asked to
self-identify potential donors. These included mothers (n=2),
daughters (n=1), sons (n=3), wives (n=1), husbands (n=1), and
friends (n=2). Prior to recruitment, the donors were required to
submit available medical records and have a separate medical
history interview away from the recipient patient. The history
included: assessment of infectious risk, including identification
of known risk factors for HIV and Hepatitis, current communicable
diseases, and recent travel to areas of the world with a higher
prevalence of diarrheal illnesses. Additional absolute donor
exclusion criteria included gastrointestinal co-morbidities and the
use of antibiotics within preceding three months. Since gut
microbiota are likely involved in various aspects of energy
metabolism and the functioning of the immune system, the presence
of features of metabolic syndrome, autoimmunity, or allergic
diseases were treated as relative exclusion criteria. Donors
provided separate informed consent to participate in the protocol,
which included risks associated with laboratory screening. The
donors underwent serologic testing for HIV and Hepatitis B and C,
and stool testing that included screening for routine enteric
pathogens, C. difficile toxin B, and examination for ova and
parasites, and Giardia and Cryptosporidium antigens.
[0150] Given varying logistic difficulties in recruiting individual
patient-identified donors, the lack of availability of donor
materials when needed, and no evidence to suggest a clear
therapeutic advantage of using a related versus unrelated donor
(e.g., son or daughter versus friend or domestic partner),
volunteer donors were recruited into the FMT program. The
advantages of this change included removing the burden of donor
identification from the patient, improving the efficiency and costs
related to donor screening, a more consistent supply donor fecal
microbiota, and the ability to impose extensive and stringent
exclusion criteria on donor selection (Table 2). Two unpaid
volunteer donors were recruited during this period, and one of them
provided the majority of donated fecal material. Donor medical
history was reviewed prior to every donation and complete
laboratory screening, as described above, was done every 6
months.
TABLE-US-00002 TABLE 2 Donor exclusion criteria. Donor Exclusion
Criteria History and Physical Examination Laboratory Screening Risk
of Infectious Agent 1. Known HIV or Hepatitis B, C infection. 1. Ab
for HIV 1 and 2. 2. Known exposure to HIV or viral 2. Viral
Hepatitis A IgM. hepatitis at any time. 3. Hepatitis B surface Ag
and core Ab. 3. High risk behaviors including sex for 4. HCV Ab.
drugs or money, men who have sex with 5. RPR. men, more than one
sexual partner in the 6. Stool cultures for enteric pathogens
preceding 12 months, history of including Salmonella, Shigella,
incarceration, any past use of Yersinia, Campylobacter, E. Colt
intravenous drugs or intranasal cocaine. 0157:H7. 4. Tattoo or body
piercing within 12 7. Ova and parasites examination. months. 8.
Positive stool Giardia, 5. Travel to areas of the world with
Cryptosporidium and Helicobacter increased risk of traveler's
diarrhea. pylori antigens. 6. Current communicable disease, e.g.,
9. Clostridium difficile toxin B PCR. upper respiratory tract viral
infection. 10. Liver function tests including alkaline phosphatase,
AST, ALT. Gastrointestinal 1. History of irritable bowel syndrome,
or comorbidities any of the associated symptoms, including frequent
abdominal cramps, excessive gas, bloating, abdominal distension,
fecal urgency, diarrhea or constipation. 2. History of inflammatory
bowel disease such as Crohn's disease, ulcerative colitis,
lymphocytic colitis. 3. Chronic diarrhea. 4. Chronic constipation
or use of laxatives. 5. History of gastrointestinal malignancy or
known colon polyposis. 6. History of any abdominal surgery, e.g.,
gastric bypass, intestinal resection, appendectomy,
cholecystectomy, etc. 7. Use of probiotics or any other over the
counter aids for specific purposes of regulating digestion.
Systemic Medical 1. Established metabolic syndrome or any 1. Serum
triglycerides (>150 mg/dL). Conditions early features suggestive
of its 2. HDL cholesterol <40 mg/dL (males) emergence. Body mass
index >26 and <50 mg/dL (females). kg/m2, waste:hip ratio
>0.85 (male) 3. High sensitivity CRP >2.4 mg/L. and >0.8
(female); BP >135 mmHg 4. Fasting plasma glucose >100
systolic and >85 mmHg diastolic. mg/dL. 2. Known systemic
autoimmunity, e.g., 5. Liver function tests, including connective
tissue disease, multiple alkaline phosphatase, AST, ALT. sclerosis,
etc. 6. FANA. 3. Known atopic diseases including asthma or eczema.
4. Chronic pain syndromes including fibromyalgia, chronic fatigue
syndrome 5. Ongoing (even if intermittent) use of any prescribed
medications, including inhalers or topical creams and ointments. 6.
Neurologic, neurodevelopmental, and neurodegenerative disorders
including autism, Parkinson's disease, etc. 7. Presence of a skin
rash, wheezing on auscultation, lymphadenopathy, hepatomegaly or
any stigmata of liver disease, swollen or tender joints, muscle
weakness, abnormal neurological examination. Additional factors 1.
Antibiotics for any indication within the known to affect the
preceding 6 months. composition of intestinal microbiota
[0151] Donor Material Preparation
[0152] Individual patient-identified donors used in the early phase
of the program came into the outpatient endoscopy center 1-2 h
prior to the scheduled procedure.
[0153] The fecal material was collected in a toilet hat and
processed in a dedicated bathroom separate from the procedure room.
Approximately 50 gm of fecal material was placed into a standard
commercial blender (Oster, Sunbeam Corp, Rye, N.Y.) and homogenized
in 250 mL of sterile, nonbacteriostatic nominal saline. The slurry
was then passed through stainless steel tea strainers to remove
larger particles that could interfere with loading the
syringes.
[0154] The material obtained from volunteer "universal" donors was
transported on ice into the laboratory, where it was processed
within two hours of collection. The material was weighed and
homogenized in a commercial blender in a dedicated biological
cabinet. The slurry was then passed through 2.0 mm, 1.0 mm, 0.5 mm,
and 0.25 mm stainless steel laboratory sieves (W. S. Tyler, Inc.,
Mentor, Ohio) to remove undigested food and smaller particulate
material. The resulting material passing through the 0.25 mm sieve
was centrifuged at 6,000.times.g for 15 min in a Sorvall SS-34
rotor and resuspended to one half the original volume in
nonbacteriostatic natural saline. The resulting concentrated fecal
bacteria suspension was administered to the patient immediately or
amended with sterile pharmaceutical grade glycerol (Sigma, St.
Louis, Mo.) to a final concentration of 10%, and stored frozen at
-80.degree. C. for one to eight weeks until used. Thawing was done
over 2-4 hours in an ice bath prior to the FMT procedure. The
frozen preparation was diluted to 250 ml with nonbacteriostatic
normal saline prior to infusion in the donor. This fecal material
extract, whether fresh or frozen, was nearly odorless and of
reduced viscosity, color, and texture relative to earlier material
prepared in the endoscopy center. Filtration of donor material
allowed for effortless loading of large tip 60 mL syringes without
risk of clogging. All containers, bottles, and sieves used in
material preparation were sterilized prior to use. Fecal material
from universal donors was treated in the same manner as that
obtained from patient-identified donors.
[0155] Transplantation Procedure
[0156] Patients were maintained on full dose of vancomycin (125 mg,
4 times daily, by mouth) until two days prior to the FMT procedure.
The day before the procedure the patients were prepped using a
split dosage polyethylene glycol purge (GoLYTELY or MoviPrep),
which is standard in our endoscopy unit, prior to colonoscopies to
wash out residual antibiotic and fecal material. The patients
underwent a full colonoscopy under conscious sedation. Mucosal
biopsies were taken to rule out lymphocytic colitis in absence of
obvious inflammatory bowel disease. The majority of the prepared
donor material (220-240 mL) was administered via the colonoscope's
biopsy channel into the patient's terminal ileum and cecum. In some
cases, however, a small portion (50 mL) was also instilled into
colonic areas containing maximal diverticulosis. Recovery procedure
was identical to that routinely used for standard colonoscopy
patients. All patients were instructed to contact the endoscopist
in case of symptom recurrence, were formally followed in clinic 1-2
months after the procedure. Clearance of CDI was defined by
resolution of diarrhea and negative stool testing for C. difficile
at 2 months following FMT. All patients in this protocol also
participated in a study examining fecal bacterial community
structure, which involved collection of fecal specimens on days 3,
7, 14 and 1, 3, 6, and 12 months after the procedure. The research
staff collected these specimens from the patient's places of
residence, providing additional opportunities for symptom
follow-up.
[0157] Statistical Analysis
[0158] Non-categorical data were compared using unpaired Student's
t-test. Categorical data were compared using Fisher's exact test.
GraphPad Prism software was used to calculate two-tailed and
two-sided p-values that were calculated with each test,
respectively.
[0159] Results
[0160] Demographics
[0161] The group of patients with recurrent CDI described in this
report clearly had refractory disease as evidenced by the average
number of sequential relapses and duration of the condition (Table
3). Furthermore, many patients had multiple risk factors for high
probability of recurrence, such as history of severe CDI as
evidenced by hospitalization, frequent use of non-C. difficile
intercurrent antibiotics, and advanced age (Hu et al.
Gastroenterology 2009; 136:1206-14). All patients failed a long
taper or pulsed regimen of vancomycin, and 40% of patients also
failed an additional long course of vancomycin followed by a
two-week rifaximin "chaser" regimen. One of these patients also
failed a 4-week course of rifaximin. Several patients (3/43) took
2-4 week course of nita7oxanide, which also failed to clear the
infection. Patients with inflammatory bowel disease were not
excluded from the protocol. Thirty five percent of our patients (14
of 40) had underlying IBD, including Crohn's disease (6/14),
ulcerative colitis (4/14), and lymphocytic colitis (4/14). The
patients with IBD were generally younger (Table 4), but did not
differ in the refractory nature of CDI or severity of presentation
than older patients. However, the majority of patients without
underlying 1BD had moderate to severe diverticulosis.
TABLE-US-00003 TABLE 3 Demographics of patient population. The
first 10 cases were done using patient-identified individual
donors. After that, the protocol shifted to use of a standard
donor. Fresh material was used in the earlier cases, and later
practice shifted to use of frozen material. Duration (months)
Number of History of Interim Donor Age Female of RCDI Relapses
Hospitaliza- Anti- Divertic- Success Material (Mean .+-. SD) Gender
(Mean .+-. SD) (Mean .+-. SD) tion for CDI biotics PPI CRI IBD
ulosis Rate Individual 61 .+-. 22 70% 12.7 .+-. 7.3 6.2 .+-. 3.0
70% 60% 60% 30% 30% 50% 7/10 Donor (70%) (n = 10) Standard 55 .+-.
22 83% 13.1 .+-. 9.8 6.4 .+-. 3.3 75% 42% 33% 25% 50% 50% 11/12
Donor, (92%) Fresh Material (n = 12) Standard 59 .+-. 21 67% 10.1
.+-. 10.0 5.2 .+-. 3.0 38% 43% 43% 14% 24% 48% 19/21 Donor, (90%)
Frozen Material (n = 12) Total 59 .+-. 21 72% 12.2 .+-. 10.3 5.9
.+-. 3.3 56% 48% 47% 21% 33% 49% 37/43 Experience (86%) RCDI =
Recurrent C. difficile Infection PPI = Proton Pump Inhibitor
medication CRI = Chronic Renal Insufficiency or Failure IBD =
Inflammatory Bowel Disease
TABLE-US-00004 TABLE 4 Comparison of patients without and with
underlying IBD. Definition of IBD includes patients with Crohn's
disease, ulcerative colitis, and incidentally discovered
lymphocytic colitis. Non-IBD (n = 29) IBD (n = 14) p Value Age
(Mean .+-. SEM) 64.7 .+-. 3.3 44.6 .+-. 5.8 p = 0.0021 Female 69%
79% p = 0.43 (NS) Duration of RCDI 13.5 .+-. 2.1 8.3 .+-. 3.3 0 =
0.09 (NS) (Mean # of months .+-. SD) Number of Relapses .+-. 6.2
.+-. 3.0 4.4 .+-. 1.3 p = 0.04 SD Rate of Hospitalization 55% 57% P
= 1.00 (NS) Interim Antibiotics 51% 36% p = 0.35 (NS) PPI 48% 43% p
= 1.00 (NS) Renal Insufficiency 32% 14% p = 0.69 NS) Diverticulosis
69% 14% p = 0.0028
[0162] Response to Treatment
[0163] The overall rate of infection clearance was 86% in response
to a single infusion of donor fecal material, as evidenced by
symptom resolution and negative PCR testing for C. difficile toxin
B after two months of follow-up (Table 3). Negative testing for C.
difficile toxin B for two months was accepted as therapeutic
success in patients with underlying IBD, even in absence of
complete symptom resolution. Three of ten patients (30%) who
received FMT using material from patient-identified individual
donors had a recurrence of CDI. Two standard donors were employed
for the remaining 33 cases in this series, but the majority (30/33)
were done using material prepared from a single donor. Three of 33
patients who received FMT from a standard donor (fresh or frozen)
had a recurrence of CDI. The difference in donor source,
patient-identified versus standard, was not significant (p=0.1270).
There was no significant difference in clearing the infection with
fresh (11/12) or frozen (19/21) donor material. All 6 patients who
experienced recurrence of CDI after FMT were offered a repeat
procedure. Two of these patients, both >80 years of age, had
multiple other active medical problems and preferred to remain on
indefinite treatment with vancomycin. Four other patients were
treated with a second infusion, and all cleared the infection
bringing the overall success rate to 95% (41 of 43 patients). All
second infusions were performed using the standard donor derived
material. One of the recurrences of CDI occurred in a patient who
received his first infusion from the second standard donor. The
same donor source was used for his second FMT. Three of the four
patients who received a second FMT had underlying IRD; two patients
had Crohn's disease and one had lymphocytic colitis. Finally, the
fourth patient had a partial colon resection done for a stricture
that developed following her initial CDI episode. She has a
colostomy draining her proximal colon and a long segment of
residual distal colon. After recurrence of CDI within three weeks
following her first FMT we thought it was likely that engraftment
in this case was complicated by difficulty in retaining the donor
material due to high flow of fecal contents and relatively small
size of the infected colon. The second infusion in this case was
done with two doses of frozen standard donor material: one via the
colostomy into the colon and the other into the jejunum using upper
push enteroscopy. C. difficile testing of her fecal material was
done weekly in the first month and monthly thereafter. No C.
difficile was found over three months of follow-up.
[0164] No serious adverse events were noted following FMT in any of
the patients, with ether fresh or frozen materials. A minority of
patients (approximately a third) noted some irregularity of bowel
movements and excessive flatulence during the first couple weeks
following the procedure, but these symptoms resolved by the time
they were seen in clinic follow-up. Enhanced colitis activity in
patients with underlying IBD was not observed and there was
improvement in overall colitis activity in all patients with UC,
although that is easily attributable to clearing the CDI.
Interestingly, all diagnoses of lymphocytic colitis were made for
the first time from biopsies taken during the colonoscopies
performed at the time of FMT. These patients completely normalized
their bowel function and had no diarrhea after FMT without any
additional medical therapy for lymphocytic colitis. Follow-up
biopsies were not performed in these patients when they became
asymptomatic.
[0165] Discussion
[0166] Recurrent infection is one of the most difficult clinical
challenges in the spectrum of C. difficile induced diarrheal
disease. The risk of recurrence increases up to 65% after two or
more episodes (McDonald et al. Emerg Infect Dis 2006; 12:409-15),
and this risk is nearly certain in older patients who suffered
severe CDI and suffered additional disruption of gut microbiota
from intercurrent administration of non-C. difficile suppressing
antibiotics (Hu et al. Gastroenterology 2009; 136:1206-14). The
inclusion criteria for patients in this case series were simple: at
least three recurrences and failure of standard antibiotic
treatments. Our patients averaged about six recurrences over an
average course of one year. This population highlights known risk
factors for recurrence of CDI other than documented recurrence. The
majority had history of at least one hospitalization for severe CDI
and almost half took antibiotics after developing CDI for another
non-C. difficile indication. Patients with inflammatory bowel
disease dominated the younger age group. Virtually all patients
were taking probiotics at presentation and many have also tried
toxin-binding resins. We did not systematically collect information
on all the various probiotics preparations taken by our patients,
and many have tried multiple types through the course of their
recurrent infections. The most common preparations contained
Saccharomyces boulardii and strains of Lactobacilli. All patients
were recommended to discontinue taking probiotics after FMT. In
summary, by all available indicators the patients in this case
series had recalcitrant CDI that would not have had a significant
response rate to a placebo, and were unlikely to respond to another
course of antibiotics or other available therapeutic options.
[0167] FMT has been used for decades as a last ditch method to cure
recurrent CDI, and there has been growing uncontrolled evidence
supporting its efficacy. Here we report one of the largest single
case series. The 95% overall success rate in this series is
comparable to the cumulative experience in the literature (Bakken.
Anaerobe 2009; 15:285-9, van Nood et al. Euro Surveill 2009; 14,
Khoruts and Sadowsky. Mucosal Immunol 2011; 4:4-7), and adds to the
impetus for developing this therapeutic approach to make it more
widely available. The major issues tackled by our center were those
of practicality. In the early phase of the program we asked the
patients to bring in prospective donors, which is the most common
approach in practice at this time. Our experience does not
contradict the efficacy of this approach. However, donor
identification and work-up increased expense of the procedure and
introduced a potential delay period. Moreover, some patients who
were already exhausted by the illness had difficulty in finding
suitable donors. While the ideal state of donor health may not be
essential for elderly recipients with limited life expectancy, we
felt compromise was not an option for younger patients on any of
the donor exclusion criteria. Gut microbiota constitute a human
microbial organ with major functions in energy metabolism and
function of the immune system (Khoruts and Sadowsky. Mucosal
Immunol 2011; 4:4-7). Therefore, this transplant procedure has
potential implications for systemic physiology of the recipient.
While donor health is not a guarantee to optimal composition of gut
microbiota, it is currently the only available indicator. For all
these reasons we decided to introduce the standard donor option to
our patients. Interestingly, although many patients came into
clinic with some potential donor already identified, they all
immediately preferred the standard option of an anonymous screened
donor upon learning about it.
[0168] The next challenge became advance preparation of the donor
material. Little is known about viability of different constituents
of fecal microbiota over time, and we did not wish to test this
variable. However, since production of fresh material on demand is
not always practical, and does create delay and issues of
sanitation and aesthetics, we introduced frozen donor material as
another treatment option. The clinical efficacy of frozen
preparation became quickly evident and it has now become part of
the standard protocol in our program.
[0169] FMT is typically considered a last choice, desperate therapy
option by most clinicians, and to a great extent that is due to
multiple aesthetic and practical barriers that stand in the way of
its administration. Increased prevalence, morbidity, and mortality
of CDI has now reached epidemic proportions and a significant
fraction of these patients cannot clear the infection with standard
therapies. These patients may benefit from FMT, but it is likely
that the procedure is not available to them. Our FMT protocol has
now progressed to the point where most obvious aesthetic and
practical challenges have been overcome. This also significantly
reduces costs associated with screening of potential donors. While
effort and organization is required for recruitment and screening
of suitable donors, as well as material preparation and banking,
execution of actual FMT has become a simple matter of loading the
syringes with thawed, nearly odorless, material and a
colonoscopy.
[0170] There are a number of limitations to this study. It was not
a rigorous clinical trial designed to test efficacy of a particular
FMT methodology versus another, or some other form of therapy.
Instead, it was an attempt to standardize FMT, as the procedure
protocol evolved in the course of our clinical experience.
Additional work is needed to ready this procedure for clinical
trials and wider application. Nevertheless, our clinical outcomes
provide very convincing evidence for efficacy of the frozen
preparations. However, we cannot conclude from this experience
alone that the fresh and frozen preparations are equivalent. The
complexity of the donor material preparations, technical inability
to culture most of the contained microbial constituents by classic
laboratory techniques, and our ignorance as to the identity of
species that are therapeutically most important precluded simple
tests of donor material prior to FMT that could predict its
efficacy. However, we are currently working to characterize the
microbial composition of donor material and recipients' fecal
samples collected over time by high throughput 16S rRNA gene
sequencing. Results of these experiments should provide some means
to compare different donor preparations. In addition, we are
working to develop practical laboratory tests that will allow for
further standardization of microbial composition of donor
preparations.
[0171] While application of FMT for recurrent CDI has a long
history, case reports suggest that it may also have a place in
treatment of IBD and IBS (Bennet et al. Lancet 1989; 1:164, Borody
et al. J Clin Gastroenterol 2003; 37:42-7, Andrews and Borody. Med
J Aust 1993; 159:633-4). Given the potentially important role of
gut microbiota in pathogenesis of the metabolic syndrome, FMT is
already being explored in a clinical trial for this condition
(Vrieze et al. Diabetologia 2010; 53:606-13). Simplification and
standardization of FMT-based therapeutics is critical for its
future development. Recent technological advances have also made it
possible to gain insight into composition of gut microbiota and
their activity. The study of microbiota in the context of FMT
should accelerate development of microbial therapeutics and yield
new insights into microbial host interactions.
[0172] The complete disclosure of all patents, patent applications,
and publications, and electronically available material (including,
for instance, nucleotide sequence submissions in, e.g., GenBank and
RefSeq, and amino acid sequence submissions in, e.g., SwissProt,
PIR, PRF, PDB, and translations from annotated coding regions in
GenBank and RefSeq) cited herein are incorporated by reference in
their entirety. Supplementary materials referenced in publications
(such as supplementary tables, supplementary figures, supplementary
materials and methods, and/or supplementary experimental data) are
likewise incorporated by reference in their entirety. In the event
that any inconsistency exists between the disclosure of the present
application and the disclosure(s) of any document incorporated
herein by reference, the disclosure of the present application
shall govern. The foregoing detailed description and examples have
been given for clarity of understanding only. No unnecessary
limitations are to be understood therefrom. The invention is not
limited to the exact details shown and described, for variations
obvious to one skilled in the art will be included within the
invention defined by the claims.
[0173] Unless otherwise indicated, all numbers expressing
quantities of components, molecular weights, and so forth used in
the specification and claims are to be understood as being modified
in all instances by the term "about." Accordingly, unless otherwise
indicated to the contrary, the numerical parameters set forth in
the specification and claims are approximations that may vary
depending upon the desired properties sought to be obtained by the
present invention. At the very least, and not as an attempt to
limit the doctrine of equivalents to the scope of the claims, each
numerical parameter should at least be construed in light of the
number of reported significant digits and by applying ordinary
rounding techniques.
[0174] Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the invention are approximations,
the numerical values set forth in the specific examples are
reported as precisely as possible. All numerical values, however,
inherently contain a range necessarily resulting from the standard
deviation found in their respective testing measurements.
[0175] All headings are for the convenience of the reader and
should not be used to limit the meaning of the text that follows
the heading, unless so specified.
* * * * *
References