U.S. patent application number 12/740139 was filed with the patent office on 2010-11-18 for composition for inhibition of transplant rejection containing the cordyceps mycellia extract as an active ingredient.
This patent application is currently assigned to HANKOOK PHARM. CO., INC.. Invention is credited to Man Woo Han, Chang-Uk Hur, Hwan-Chul Kim, Jin Pyo Kim, Jae Kuk Yoo.
Application Number | 20100291154 12/740139 |
Document ID | / |
Family ID | 40717857 |
Filed Date | 2010-11-18 |
United States Patent
Application |
20100291154 |
Kind Code |
A1 |
Han; Man Woo ; et
al. |
November 18, 2010 |
COMPOSITION FOR INHIBITION OF TRANSPLANT REJECTION CONTAINING THE
CORDYCEPS MYCELLIA EXTRACT AS AN ACTIVE INGREDIENT
Abstract
Disclosed is a composition for the inhibition of transplant
rejection and the prevention and treatment of skin diseases,
comprising a cordyceps mycellia extract as an active ingredient.
The cordyceps mycellia extract significantly suppresses the
production of antibodies to transplants without side effects, such
as weight change. Based on natural material, the composition is
non-toxic and harmless to the human body and thus can be used as an
immunosuppressant for organ transplantation. Also, it stops oozing
from sores and is useful in the prevention and treatment of skin
diseases, including atopy, allergic reactions, decubitus ulcers,
pemphigus and smallpox.
Inventors: |
Han; Man Woo; (Daejeon,
KR) ; Yoo; Jae Kuk; (Daejeon, KR) ; Hur;
Chang-Uk; (Daejeon, KR) ; Kim; Hwan-Chul;
(Daejeon, KR) ; Kim; Jin Pyo; (Daejeon,
KR) |
Correspondence
Address: |
LUCAS & MERCANTI, LLP
475 PARK AVENUE SOUTH, 15TH FLOOR
NEW YORK
NY
10016
US
|
Assignee: |
HANKOOK PHARM. CO., INC.
Nonsan-si, Chungcheongnam-do
KR
|
Family ID: |
40717857 |
Appl. No.: |
12/740139 |
Filed: |
December 7, 2007 |
PCT Filed: |
December 7, 2007 |
PCT NO: |
PCT/KR2007/006356 |
371 Date: |
April 28, 2010 |
Current U.S.
Class: |
424/274.1 ;
424/195.15 |
Current CPC
Class: |
A61P 17/02 20180101;
A61P 31/20 20180101; A61P 37/08 20180101; A61K 36/068 20130101;
A61P 37/00 20180101; A61P 17/00 20180101; A61P 37/06 20180101 |
Class at
Publication: |
424/274.1 ;
424/195.15 |
International
Class: |
A61K 36/068 20060101
A61K036/068; A61K 36/06 20060101 A61K036/06; A61P 37/06 20060101
A61P037/06; A61P 17/00 20060101 A61P017/00; A61P 17/02 20060101
A61P017/02; A61P 31/20 20060101 A61P031/20 |
Claims
1. An immunosuppressive composition for inhibition of transplant
injection, comprising a mycelial extract from vegetable worm as an
active ingredient.
2. The immunosuppressive composition according to claim 1, wherein
the vegetable worm is one selected from a group consisting of
Cordyceps militaris, Cordyceps sinensis, Hymenostilbe odonatae,
Cordyceps nutans, Tilachlidiopsis nigra, Paecilomyces japonica,
Cordyceps tricentri and Cordyceps sphecocephala.
3. The immunosuppressive composition according to claim 1, wherein
the composition is in a dosage form suitable for oral, intravenous
or abdominal administration.
4. The immunosuppressive composition according to claim 3, wherein
the composition is orally administered at a dose from 550 to 2,200
mg/day.
5. The immunosuppressive composition according to claim 1, wherein
the composition suppresses production of antibodies after organ or
tissue transplantation, said antibodies causing transplant
rejection.
6. A composition for prevention and treatment of skin diseases,
comprising a cordyceps mycellia extract as an active
ingredient.
7. The composition according to claim 6, wherein the skin diseases
are atopy, decubitus ulcer, pemphigus and/or smallpox.
Description
TECHNICAL FIELD
[0001] The present invention relates to a composition for
inhibition of transplant rejection, comprising a cordyceps mycellia
extract as an active ingredient.
BACKGROUND ART
[0002] Transplant rejection occurs when the immune system of the
recipient of a transplant attacks a transplanted organ or tissue.
Thus, effective suppression of the immune response is known as a
main factor determining the success of transplantation. In this
regard, the development of immunosuppressive medications has
brought about exceptional advances in the transplantation of organs
and tissues and the treatment of autoimmune diseases and has made a
great contribution to the study of the in vivo mechanism of immune
responses to the transplanted organ or tissue.
[0003] As described, immunosuppressive drugs were developed to
inhibit or attenuate transplant rejection. An example is
cyclosporine A (U.S. Pat. No. 4,117,118) produced from
Tolypocladium inflatum, a soil fungus. These immunosuppressive
drugs not only help realize clinically successful organ
transplantation, but also suggest the therapeutic use thereof in
treating autoimmune diseases. Even though they are required to act
selectively and specifically for T-cells only, conventional
immunosuppressive drugs have an influence on a wide range of
cellular functions, including general signal pathways, causing side
effects on other organs, which are healthy (see. S.-H. Lee et al.,
Korean J. Immunology, 19:375.about.389 (1997)). For instance,
cyclosporine A is known to show side effects of chronic liver
diseases and hypertension after heart transplantation (see: J. E.
F. Reynolds, et al., Martindale The Extra Pharmacopoeia, 31.sup.st
ed., pp. 557-562, Royal Pharmaceutical Society, London, 1996). Many
attempts have been made to develop novel immunosuppressive drugs
free of side effects. FK-506 has recently been discovered to be an
immunosuppressant, and has been commercialized. However, side
effects of this drug have also been found (Clin. Transplantation,
11: 237.about.242 (1997)).
[0004] In China, vegetable worms, together with Korean ginseng,
have long been used as precious materials in medicinal cuisine for
special people in the aristocratic classes. Vegetable worms are a
kind of medicinal fungus produced as a result of the parasitism of
vegetable worms on insects. In high temperature and moisture
conditions, the vegetable worms, which are actually fungus, infect
living insects, proliferate therein to kill the host insects, and
form fruiting bodies on the surface of the host insects. As used
herein, the term "vegetable worm", is intended to primarily refer
to Cordyceps sinensis, a parasite on larvae of the Hepialidae
family, but at present generally refers to all fungi attacking
arthropods, such as spiders.
[0005] Cordyceps sinensis breaks down into 10.8% water, 8.4%
lipids, 25.about.32% crude proteins, 23.9% carbohydrates, and 18.5%
crude fibers. In this vegetable worm are found 17 different amino
acids, including 8 essential amino acids. Also, it contains a trace
amount of cordycepin, 7.6% of D-mannitol and 11.2% of
polysaccharides, all known as medicinally active materials.
Cordycepin, a derivative of the nucleoside adenosine, is an isomer
of quinic acid, known to show anti-cancer activity.
[0006] Various medicinally valuable activities of extracts from
vegetable worms discovered thus far include antibacterial activity
(Staphylococcus, Streptococcus, Bacterium mallei, Bacillus
anthracis, Pasteurella suiseptica, Microsporum gypseum, and
Microsporum lanosum), activity on the central nervous system
(sedative, anticonvulsant activity), the respiratory system
(bronchial asthma healing, expectorant activity) and the
cardiovascular system (stabilization of heart beats, reduction of
cholesterol level, anti-hypoxia activity), anticancer activity,
immuno potentiation, anti-fatigue activity, and anti-aging
activity.
[0007] However, nowhere has the use of vegetable worm extracts as
an immunosuppressant been disclosed in the prior art.
[0008] Leading to the present invention, intensive and thorough
research on an immunosuppressant entailing no side effects,
conducted by the present inventors, resulted in the finding that an
extract from vegetable worm mycelia significantly inhibits the
immune response to transplanted organs or tissues.
DISCLOSURE
Technical Problem
[0009] It is therefore an object of the present invention to
provide an immunosuppressive composition, useful in the prevention
of transplant rejection, comprising a cordyceps mycellia extract as
an active ingredient.
[0010] It is another object of the present invention to provide a
composition for the prevention and treatment of skin diseases,
comprising a cordyceps mycellia extract as an active
ingredient.
Technical Solution
[0011] In order to accomplish the objects of the present invention,
an immunosuppressive composition comprising a mycelial extract from
a vegetable worm as an active ingredient is provided for the
inhibition of transplant injection.
[0012] Also, a composition comprising a cordyceps mycellia extract
as an active ingredient is provided for the prevention and
treatment of skin diseases.
ADVANTAGEOUS EFFECTS
[0013] The cordyceps mycellia extract was found to significantly
suppress the production of antibodies to transplants without side
effects, such as weight change. Based on a natural material, the
composition is non-toxic and harmless to the human body, and thus
can be used as an immunosuppressant for organ transplantation.
Also, it arrests oozing from sores and is applicable to the
prevention and treatment of skin diseases, including atopy,
allergic reactions, decubitus ulcers, pemphigus and smallpox.
DESCRIPTION OF DRAWINGS
[0014] FIG. 1 is a graph showing the levels of antibodies produced
against splenocytes transplanted into mice administered with the
cordyceps mycellia extract of the present invention or with saline
in accordance with an embodiment of the present invention
(.box-solid.: control, .DELTA.: experimental group).
[0015] FIG. 2 is a graph showing changes in the weight of the mice
administered with the cordyceps mycellia extract of the present
invention and with saline in accordance with an embodiment of the
present invention (.box-solid.: control, .DELTA.: experimental
group).
[0016] FIG. 3 shows the dermal states of mice administered with the
cordyceps mycellia extract of the present invention and with saline
in photographs (panel a: experimental group, panel b: control).
[0017] FIG. 4 is a graph showing the levels of antibodies produced
against splenocytes transplanted into mice administered with the
cordyceps mycellia extract of the present invention or with other
comparative chemicals in accordance with another embodiment of the
present invention.
[0018] FIG. 5 is a graph showing changes in the weight of the mice
administered with the cordyceps mycellia extract of the present
invention and with other comparative chemicals in accordance with
another embodiment of the present invention.
BEST MODE
[0019] In accordance with an aspect of the present invention, an
immunosuppressive composition based on a cordyceps mycellia extract
is provided for the inhibition of transplant rejection.
[0020] The cordyceps mycellia extract may be obtained from cultured
mycelia, or may be commercially available. For example, vegetable
worm powder, which is sold as a health aid food, may be used in the
present invention, whether it comes from fruit bodies, mycelia, or
a combination thereof.
[0021] Examples of the vegetable worm useful in the present
invention include Cordyceps militaris, Cordyceps sinensis, which is
parasitic on larvae of the Hepialidae family, Hymenostilbe
odonatae, Cordyceps nutans, Tilachlidiopsis nigra, Paecilomyces
japonica, Cordyceps tricentri, and Cordyceps sphecocephala.
[0022] The cordyceps mycellia extract according to the present
invention was tested for immunosuppressive effect on smallpox mouse
models. After the transplantation of splenocytes thereinto, the
animal models were administered with the cordyceps mycellia
extract. ELISA analysis on the animal models for the quantification
of antibodies to the splenocytes indicated that the cordyceps
mycellia extract of the present invention significantly inhibits
the production of the antibodies (see FIGS. 1 and 4). Therefore,
the cordyceps mycellia extract according to the present invention
can be used as an immunosuppressant for the prevention of
transplant rejection.
[0023] Also, observations were made of whether the cordyceps
mycellia extract causes side effects in vivo. Almost no changes
were found in the weight of the mice after the transplantation of
splenocytes (see FIGS. 2 and 5). Hence, the cordyceps mycellia
extract can be used as an immunosuppressant for the prevention of
transplant rejection without the occurrence of side effects, such
as changes in weight, upon organ transplantation.
[0024] In accordance with another aspect thereof, the present
invention provides a composition for the prevention and treatment
of skin diseases, comprising a cordyceps mycellia extract as an
active ingredient.
[0025] The cordyceps mycellia extract according to the present
invention was tested for therapeutic activity for skin diseases on
smallpox mouse models. After the administration of the cordyceps
mycellia extract thereinto, the smallpox mouse models were observed
to stop oozing from the smallpox sores (FIG. 3). In contrast, the
control, which was not administered with the cordyceps mycellia
extract of the present invention, was observed to ooze from the
smallpox sores and remain depilated at the sores. Hence, the
cordyceps mycellia extract according to the present invention can
be used for the treatment of skin diseases, such as atopy, allergic
reactions, decubitus ulcers, pemphigus, smallpox, etc.
[0026] When used as medications or health aid foods, the
composition may further comprise one or more active ingredients
having a function similar to that of the cordyceps mycellia
extract.
[0027] The cordyceps mycellia extract in accordance with the
present invention can be administered orally or non-orally, and may
be provided in general medicinal forms. For clinical practice, the
cordyceps mycellia extract of the present invention may be used in
oral or non-oral forms. It is usually formulated in combination
with a diluent or an excipient, such as a filler, a thickening
agent, a binder, a wetting agent, a disintegrant, a surfactant,
etc. Solid agents intended for oral administration of the extract
of the present invention may be in the form of tablets, pills,
powders, granules, capsules, and the like. In these solid agents,
the cordyceps mycellia extract of the present invention is
formulated in combination with at least one excipient, such as
dextrin, starch, calcium carbonate, sucrose, lactose, or gelatine.
In addition, a lubricant, such as magnesium stearate, talc, or the
like, may also be added. Liquid agents intended for oral
administration include suspensions, internal use solutions,
emulsion, syrups, and the like. In addition to a simple diluent,
such as water or liquid paraffin, various excipients, such as
wetting agents, sweetening agents, aromatics, preservatives, and
the like, may be contained in the liquid agents for the oral
administration of the extract of the present invention. Also,
non-oral dosage forms of the extract of the present invention
include sterile aqueous solutions, non-aqueous solutions,
suspensions and emulsions, freeze-dried agents, and suppositories.
Non-aqueous solutions and suspensions made from propylene glycol,
polyethylene glycol, vegetable oils, such as olive oil, and
injectable esters such as ethyl oleate may be used. The basic
materials of suppositories include Witepsol, macrogol, Tween 61,
cacao butter, laurin, glycerol, and gelatin.
[0028] The effective dosage of the cordyceps mycellia extract in
accordance with the present invention depends on various factors,
including the patient's weight, age, gender, state of health, diet,
the time of administration, route of administration, excretion
rate, etc. For oral administration, the cordyceps mycellia extract
in accordance with the present invention may be administered at a
dose ranging from 550 to 2,200 mg/day.
[0029] For application to the treatment of skin diseases, the
cordyceps mycellia extract of the present invention may be used
alone or in combination with other therapies, including surgery,
radiotherapy, hormonal therapy, chemical therapy and/or biological
reaction regulators.
MODE FOR INVENTION
[0030] A better understanding of the present invention may be
obtained in light of the following examples, which are set forth to
illustrate, but are not to be construed to limit the present
invention.
Example 1
Immunosuppression Assay on Smallpox Mouse Model
[0031] An immunosuppression test was conducted with the cordyceps
mycellia extract of the present invention on smallpox mouse models
(obtained from Microbiology.cndot.Immunology Lab of the Medical
College in Keio Univ.).
[0032] From two days before the transplantation of splenocytes
(Dsg3-/-), five smallpox mice were administered orally with a
cordyceps mycellia extract powder (Cordyma.RTM.; Han Kook Sin Yak)
at a dose of 10 mg/kg/day. For 35 days (5 weeks) after the
splenocyte transplantation, the cordyceps mycellia extract was
orally administered at a dose of 10 mg/kg/day. For a control,
physiological saline was used instead of the extract.
[0033] Blood samples were taken from the mice 7 days (1 week), 14
days (2 weeks), 21 days (3 weeks), 28 days (4 weeks) and 35 days (5
weeks) after the transplantation, and were analyzed using ELISA to
determine the level of antibodies to the splenocytes. Throughout
the experiment, the weights and skin states of the mice were
monitored every day.
[0034] The results are depicted in FIGS. 1 to 3.
[0035] In FIG. 1, the levels of antibodies to the splenocytes are
plotted against time for the experimental group and the control
group. FIG. 2 shows changes in weight for the experimental group
and the control group. In FIG. 3, the experimental group and the
control group are compared with each other with respect to skin
state.
[0036] As seen in FIG. 1, the level of antibodies to the
transplanted splenocytes in the mice administered with the
cordyceps mycellia extract of the present invention was almost
zero. From this, it is apparent that the cordyceps mycellia extract
according to the present invention effectively inhibits the
production of antibodies to transplants.
[0037] In FIG. 2, it can be observed that the weight of the control
administered with physiological saline slightly increased
immediately after the transplantation, but sharply decreased from 7
days after the transplantation, as transplant rejection occurred.
In contrast, almost no change was found in the weight of the mice
administered with the cordyceps mycellia extract of the present
invention. Demonstrated to effectively inhibit the transplant
rejection and cause no side effects, such as weight gain, the
composition comprising the cordyceps mycellia extract according to
the present invention can therefore be used as an immunosuppressive
medication applicable for organ or tissue transplantation.
[0038] Furthermore, as shown in FIG. 3, while the control suffered
from sores due to smallpox, skin disease was suppressed in the mice
administered with the cordyceps mycellia extract of the present
invention. Hence, the cordyceps mycellia extract according to the
present invention can be applied to the treatment of skin diseases
including atopy, allergic reactions, decubitus ulcers, pemphigus,
smallpox, etc.
Example 1
Immunosuppression Assay on Pemphigus Mouse Model
[0039] An immunosuppression test was conducted with the cordyceps
mycellia extract of the present invention on pemphigus mouse
models, as follows.
[0040] Two S129 Dsg3-/- mice and 24 S129 Rag2-/- mice were prepared
as transplantation donors and recipients, respectively. The
recipient mice were divided into four groups of six: control (CMC
administered), comparative group (cyclophosphamide (CPA)
administered), experimental group 1 and experimental group 2
(cordyceps mycellia extract (Cordyma.RTM.; Han Kook Sin Yak)
administered). The transplantation donor was subjected to an immune
reaction by administering a Dsg3-His protein thereto, as described
below. First, 10 .mu.g of mouse Dsg3-His protein was emulsified
with the same amount of complete Freund's adjuvant (CFA) and
subcutaneously injected into the mice (1.sup.st). One week after
the first immunization, an emulsion of 10 .mu.g of mouse Dsg3-His
protein in the same amount of incomplete Freund's adjuvant was
subcutaneously injected (2.sup.nd). One week after the 2.sup.nd
immunization, subcutaneous injection was carried out in the same
manner as in the second immunization (3.sup.rd) One week later, 10
.mu.g of the protein was intraperitoneally administered (4.sup.th).
One week after the 4.sup.th immunization, intraperitoneal injection
was performed in the same manner as in the 4.sup.th immunization
(5.sup.th). Three days before transplantation, intraperitoneal
injection was performed in the same manner as in the 4.sup.th
immunization.
[0041] From one day before the transplantation of splenocytes
(Dsg3-/-), oral administration was conducted with 1 ml of the
cordyceps mycellia extract (Cordyma.RTM.; Han Kook Sin Yak) for
each experimental group, 1 ml of CMC for the control group and 1 ml
of cyclophosphamide for the comparative group. Thereafter,
splenocytes were transplanted at a density of 1.5.times.10.sup.6
cells/500 .mu.l into each mouse, followed by oral administration of
1 ml of the test material to the mice on day zero, 1, 4, 7, 11, 14,
18, 21, 25, and 28 after the transplantation. Afterwards, blood
samples were taken from the mice on day zero, 1, 4, 7, 11, 14, 18,
21, 25 and 28 after the transplantation, and were analyzed to
determine the level of antibodies to the splenocytes using ELISA.
Throughout the experiment, the weights and skin states of the mice
were monitored every day.
[0042] The cordyceps mycellia extract (Cordyma.RTM.; Han Kook Sin
Yak) for the experimental group 1 was prepared by placing 500 mg of
a powder of Isaria japonica in a 15 ml tube, adding a 0.5% CM-Na
solution (hereinafter referred to as "CMC") to the tube to form a
total volume of 5 ml, and sonicating it for 30 min in a bath to
form a suspension (dosage: 2000 mg/kg/administration). As for the
experimental group 2, its cordyceps mycellia extract (Cordyma.RTM.;
Han Kook Sin Yak) was prepared by mixing 0.5 ml of the cordyceps
mycellia extract (Cordyma.RTM.; Han Kook Sin Yak) for the
experimental group 1 with 4.5 ml of CMC and treating the mixture
for 30 min in a sonication bath to give a suspension (dosage: 200
mg/kg/administration).
[0043] Also, the cyclophosphamide administered to the comparative
group was a suspension prepared by pulverizing 50 mg of the Endoxan
P tablet, commercially available from Shionogi Pharmaceutical
Corporation, mixing the powder with 16.7 ml of CMC, and sonicating
the mixture in a bath for 30 min (dosage: 60
mg/kg/administration).
[0044] Their dermal conditions were examined on the abdominal side,
the dorsal side, the right side, and the left side for blistering,
depilation and swelling with the naked eyes while pictures were
taken of the entire dorsal side and three facial sides (right, left
and chin) with a digital camera for more precise monitoring.
[0045] The results are shown in FIGS. 4 and 5.
[0046] In FIG. 4, the levels of the antibodies to the splenocytes
were plotted against time for the experimental groups, the control
group and the comparative group. FIG. 5 shows changes in weight for
the experimental groups, the control group and the comparative
group.
[0047] As seen in FIG. 4, the production level of antibodies to the
transplanted splenocytes was observed to be lower in the mice
administered with the cordyceps mycellia extract of the present
invention than in the mice administered with CMC only (control). As
for ELISA values, a Dunnett's multiple comparison test was
performed on the basis of a PBS group. On Day 11 after the
transplantation, there was significance for the experimental group
1 (p<0.05), but not significance for the experimental group 2.
On Day 14, there was significance for both the experimental group 1
(p<0.01) and the experimental group 2 (p<0.05). However, no
significance was observed for either of them from Day 18.
Accordingly, the significant difference on Days 11 and 14 between
the experimental group administered with the cordyceps mycellia
extract at a dose of 2000 mg/kg and the control group indicates
that the cordyceps mycellia extract of the present invention
significantly inhibits the production of antibodies responsible for
transplant rejection.
[0048] For weight change, a Dunnett's multiple comparison test was
performed on the basis of the control group. On Day 11 after the
transplantation, as shown in FIG. 5, there was significance for the
experimental group 1 (p<0.05), but not significance for the
experimental group 2. On Day 14, there was significance for both
the experimental group 1 (p<0.05) and the experimental group 2
(p<0.05). However, no significance was observed for either of
them from Day 18. Accordingly, these results indicate that the
cordyceps mycellia extract of the present invention was found to
effectively suppress transplant rejection, as demonstrated by the
significant difference on Days 11 and 14 between the experimental
group administered with the cordyceps mycellia extract at a dose of
2000 mg/kg and the control group.
[0049] Furthermore, while the control suffered from skin diseases,
such as sores and depilation due to pemphigus, the skin diseases
were suppressed in the mice administered with the cordyceps
mycellia extract of the present invention. Hence, the cordyceps
mycellia extract according to the present invention can be applied
to the treatment of skin diseases including atopy, allergic
reactions, decubitus ulcers, pemphigus, smallpox, etc.
[0050] The composition of the present invention can be prepared as
described below.
Preparation Example 1
Preparation of Pharmaceutical Formulations
[0051] 1-1. Preparation of Powder
TABLE-US-00001 Cordyceps mycellia extract 1 g Dextrin 0.1 g
[0052] The above ingredients were mixed and loaded into an airtight
sac to produce powder.
[0053] 1-2. Preparation of Tablet
TABLE-US-00002 Cordyceps mycellia extract 500 mg Dextrin 45 mg Mg
Stearate 5 mg
[0054] These ingredients were mixed and prepared into tablets using
a typical tabletting method.
[0055] 1-3. Preparation of Capsule
TABLE-US-00003 Cordyceps mycellia extract 500 mg Dextrin 50 mg
[0056] These ingredients were mixed and loaded into gelatin
capsules according to a typical method to produce capsules.
[0057] 1-4. Preparation of Injection
TABLE-US-00004 Cordyceps mycellia extract 50 mg/ml Diluted HCl BP
added to form pH 3.5 NaCl BP injection up to 1 ml
[0058] The cordyceps mycellia extract was dissolved in a suitable
volume of an NaCl BP injection, and the solution was adjusted to a
pH of 3.5 with diluted HCl BP and to a desired volume with NaCl BP
injection, followed by sufficient mixing. The solution was loaded
into transparent ml type I ampules, which were hermetically sealed
by melting, followed by autoclaving at 120.degree. C. for 15 min to
prepare injections.
* * * * *