Improved oral cyclosporin formulations which have high bioavailability and are capable of administration in hard capsules, are provided. In the subject formulations, cyclosporin is delivered in an orally acceptable vehicle comprising at least one alkanol of from 2 to 3 carbon atoms in combination with at least one non-ionic surfactant and an ester of an alcohol and a fatty acid having a hydrocarbon chain of from 14 to 18 carbon atoms. The subject formulations find use in immunosuppressive therapy.

FIELD OF THE INVENTION 
The field of this invention is oral cyclosporin formulations. 
BACKGROUND 
Despite efforts to avoid graft rejection through host-donor tissue type 
matching, in the majority of transplantation procedures where a donor 
organ is introduced into a host, immunosuppressive therapy is critical to 
the maintained viability of the donor organ in the host. A variety of 
immunosuppressive agents have been employed in transplantation procedures, 
including azathioprine, methotrexate, cyclophosphamide, FK-506, rapamycin 
and corticosteroids. Agents finding increasing use in immunosuppressive 
therapy due to their preferential effect on T-cell mediated reactions are 
the cyclosporins. 
Cyclosporins are a class of cyclic polypeptides consisting of eleven amino 
acids which are produced as a metabolite by the fungus species 
Tolypocladium inflatum Gams. Cyclosporins have been observed to reversibly 
inhibit immunocompetent lymphocytes, particularly T-lymphocytes, in the 
G.sub.0 or G.sub.1 phase of the cell cycle. Cyclosporins have also been 
observed to reversibly inhibit lymphokine production and release. Although 
a number of cyclosporins are known, Cyclosporin A is the most widely used. 
Use of Cyclosporin A has been reported to prolong the survival of 
allogeneic transplants involving skin, heart, kidney, pancreas, bone 
marrow, small intestine and lung. In allogeneic transplantations, 
Cyclosporin A has been shown to suppress humoral immunity and, to a 
greater extent, cell mediated immune reactions, including: allograft 
rejection, delayed hypersensitivity, experimental allergic 
encephalomyelitis, Freund's adjuvant arthritis, and graft vs. host 
disease. Although success has been realized with Cyclosporin A, following 
transplantation administration of the agent must be continued since the 
benefits of cyclosporin therapy are reversible and graft rejection occurs 
once administration of Cyclosporin A is discontinued. 
Although cyclosporin formulations for both oral and intravenous 
administration have been developed, because of the ease of administration 
and greater patient acceptance, oral administration of cyclosporin is 
preferred. Furthermore, intravenous administration of cyclosporin can 
result in anaphylactic reactions, a side effect not observed with oral 
formulations. Oral cyclosporin formulations which have been developed and 
are currently marketed include both soft gelatin capsule and solution 
formulations, both of which are sold under the trademark SANDIMMUNE.RTM.. 
In using oral cyclosporin formulations in immunosuppressive therapy, both 
the care giver and manufacturer must be cognizant of many issues. With 
oral cyclosporin formulations, cyclosporin bioavailability can be limited 
because of cyclosporin's immiscibility in water and the tendency of 
cyclosporin to precipitate in aqueous environments. In addition, the 
concentration of cyclosporin present in oral formulations can be limited 
due to cyclosporin's hydrophobic nature. Furthermore, cyclosporin 
absorption by the gastrointestinal tract can be erratic from one 
formulation batch to the next, requiring constant monitoring of 
cyclosporin blood levels during treatment. Finally, packaging and storage 
stability are an issue with oral formulations. For example, with soft 
gelatin capsule formulations of cyclosporin, air tight packaging must be 
employed, which is inconvenient due to bulkiness and high cost. 
Thus, desirable oral cyclosporin formulations would be formulations that 
address at least some of the above issues. Ideally, oral formulations 
would promote high bioavailability, comprise high concentrations of 
cyclosporin and would be amenable to preparation in hard capsule form. 
Relevant Literature 
Physician's Desk Reference (1994) pp 2071-2074 describes oral cyclosporin 
formulations currently sold under the trademark SANDIMMUNE.RTM.. 
U.S. Patents of interest describing cyclosporins and derivatives thereof 
include: U.S. Pat. Nos. 4,220,641; 4,639,434; 4,289,851; and 4,384,996. 
U.S. Pat. No. 5,047,396 describes an intravenous preparation for 
administration of cyclosporin. U.S. Pat. Nos. 4,388,307; 4,970,076 and 
4,990,337 describe the preparation of oral cyclosporin formulations. 
The preparation of hard capsules for the oral delivery of pharmaceutical 
formulations is described in U.S. Pat. Nos. 4,822,618; 4,576,284; 
5,120,710; and 4,894,235. 
SUMMARY OF THE INVENTION 
Oral cyclosporin formulations, and methods for their use in 
immunosuppressive therapy, are provided. The subject formulations comprise 
cyclosporin in an orally acceptable vehicle comprising at least one 
alkanol of from 2 to 3 carbon atoms and at least one non-ionic surfactant 
in combination with fatty acid esters in which the acyl group contains a 
hydrocarbon chain of from 14 to 18 carbon atoms. The cyclosporin 
formulations can be packaged as hard capsules.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS 
Oral cyclosporin formulations are provided which can be formulated as 
capsules, particularly hard capsules, and promote bioavailability. In the 
subject formulations, cyclosporin is present in an orally acceptable 
vehicle comprising at least one alkanol of from 2 to 3 carbon atoms in 
combination, at least one non-ionic surfactant, and an ester of a fatty 
acid having a hydrocarbon chain of from 14 to 18 carbon atoms. In addition 
to providing for high bioavailability, the subject formulations provide 
for reproducible cyclosporin absorption from one batch of a formulation to 
the next. The subject formulations find use in immunosuppressive therapy. 
A number of cyclosporins are known in the art to exhibit immunosuppressive 
activity and may be delivered in the subject oral formulations. 
Cyclosporins that may be administered in the subject formulations include 
Cyclosporin A, Cyclosporin B, Cyclosporin C, Cyclosporin D and Cyclosporin 
G, as well as synthetic analogs thereof See Merck Index (1989) 2759. The 
subject oral formulations are particularly suited for the delivery of 
Cyclosporin A. When delivered in the subject formulations, Cyclosporin A 
will be present in concentrations ranging from 50 to 150 mg/ml, usually 
100 to 150 mg/ml, based on the volume of the vehicle component of the 
formulation. 
The vehicle component of the subject formulation will include at least one 
alkanol, usually not more than two alkanols, where the alkanol will 
usually be from 2 to 3 carbon atoms, and have not more than 1 hydroxy 
group per 1.5 carbon atoms. The alkanol may have 1 to 2 hydroxyl groups. 
Suitable alkanols include ethanol and propylene glycol. The total amount 
of alkanol in the formulation will range from 5 to 35%, usually from about 
5 to 30% (v/v) of the formulation. When present, the amount of ethanol in 
the subject formulation will range from 5 to 15%, usually from about 5 to 
10% (v/v) of the formulation. The amount of propylene glycol, when 
present, will range from 5 to 35%, usually from about 10 to 30% (v/v) of 
the formulation. 
Also present in the orally acceptable vehicle will be at least one 
non-ionic polyoxyalkylene surfactant, usually not more than two 
polyoxyalkylene non-ionic surfactants. The polyoxyalkylene surfactants 
will have a hydrophilic-lipophilic-balance (HLB) of from about 5 to 20, 
usually from about 8 to 16. Preferably, the polyoxyalkylene non-ionic 
surfactants employed in the subject formulations will be polyoxyethylene 
compounds. Polyoxyethylene compounds include: ethoxlyated alcohols, i.e. 
polyoxyethylene alcohols or ethoxylated fatty alcohols, generally of from 
10 to 18, usually from 10 to 14 carbon atoms, as well as ether and ester 
substituents thereof; and fatty acid monoesters of ethoxylated polyols of 
from 4 to 6 carbon atoms, usually 6 carbon atoms, e.g. sorbitol. The 
number of ethylenoxy groups will generally be in the range of 2 to 30, 
usually in the range from about 2 to 25. Preferred surfactants are 
polyoxyethylene (4) lauryl ether (BRIJ 30.RTM.) and polyoxyethylene (20) 
mono sorbitan mono-oleate (TWEEN 80.RTM.). The total amount of non-ionic 
surfactants present in the subject formulations will range from 5 to 50%, 
usually from about 10 to 40% (v/v) of the formulation. Where TWEEN 80.RTM. 
is present in the formulation, it will usually be present in amounts 
ranging from 5 to 50%, more usually from about 20 to 30% (v/v) of the 
formulation. When BRU 30.RTM. is present in the subject formulation, it 
will usually be present in amounts ranging from 10 to 45%, more usually 
from about 15 to 40% (v/v) of the formulation. 
In the subject formulations, in combination with the alkanol and non-ionic 
surfactant will be an ester of a fatty acid, where the hydrocarbon chain 
of the fatty acid will be from 14 to 18 carbon atoms in length and will 
generally be an even numbered chain, where the hydrocarbon chain may be 
saturated or unsaturated, usually of not more than two sites of 
unsaturation. Fatty acids of interest will generally be of plant or 
mammalian origin and include palmitate, stearate, palmitoleate, linoleate, 
linolineate and the like, particularly myristate and oleate. The alcohol 
of the fatty acid ester will be a lower alkanol of from 2 to 4 carbon 
atoms in length, usually 2 to 3 carbon atoms in length, with or without 
branches. Fatty acid esters of particular interest are isopropyl myristate 
and ethyl oleate. The fatty acid ester component of the subject 
formulations will make up from about 35 to 80% (v/v) of the formulation, 
where isopropyl myristate, when present, will range from about 55 to 75% 
(v/v), and ethyl oleate, when present, will range from about 35 to 75% 
(v/v) of the total formulation. Usually the fatty acid ester will be 
present in an amount at least about equal (v/v) and up to 8 times the 
amount of surfactant in the formulation, usually not greater than 5 times 
the amount of surfactant in the formulation (v/v). 
Optionally, the subject formulations may comprise a polyethylene glycol, 
usually polyethylene glycol 200 to 400. Polyethylene glycol, when included 
in the subject formulations, will be present in amounts ranging from 5 to 
15%, usually 5 to 10% (v/v) of the formulation. 
In some instances, it is found that an ester of a fatty acid is not 
essential to the formulation. In such instances, the formulation will 
comprise ethanol and propylene glycol in combination with a 
polyoxyalkylene non-ionic surfactant. In such formulations, the amount of 
ethanol will range from about 5 to 15% (v/v), usually from about 5 to 10% 
(v/v), while the amount of propylene glycol will range from about 40 to 
55% (v/v), usually from about 45 to 50% of the formulation. The 
formulation will comprise at least one non-ionic polyoxyethylene 
surfactant, wherein said surfactant is selected from the group consisting 
of polyoxyethylene ethers and fatty acid monoesters of ethoxylated polyols 
of from 4 to 6 carbon atoms, e.g. polyoxyethylene (20) mono sorbitan 
mono-oleate, and will be present in said formulation in an amount ranging 
from 40 to 55% (v/v), usually from about 45 to 50% (v/v) of the 
formulation. 
Of particular interest are formulations in which the non-ionic surfactant 
is a fatty acid monoester of sorbitol, where the ratio of fatty acid ester 
to surfactant in the formulation ranges from about 1.5 to 2.5, and is 
usually about 2. Formulations which are less preferred are those 
formulations comprising polyoxyethylene (4) lauryl ether as the non-ionic 
surfactant and isopropyl myristate as the fatty acid ester, where the 
ratio of fatty acid ester to surfactant in the formulation is in excess of 
about 1.5. 
Also present in the subject formulations may be a number of minor 
components which provide various functions, such as enzyme inhibitors, 
preservatives, antioxidants, antimicrobial agents, stabilizers and the 
like. The total amount of these additives, when present in the 
formulation, will normally not be greater than 5 weight % of the 
formulation. A number of excipients may also be present in the subject 
formulations, as is known in the art. 
The subject formulations are suitable for administration in capsule form, 
e.g. hard and soft capsules. Methods of producing hard capsules comprising 
liquid formulations are known in the art and described in U.S. Pat. Nos. 
4,822,618 and 4,576,284, the disclosures of which are herein incorporated 
by reference. Generally, hard capsules that find use with the subject 
formulations will comprise two parts: a shell component and a cap 
component. The shell and cap components fit together to produce an 
enclosed cavity of defined volume sealed in a hard capsule shell. The 
shell and cap components may be fabricated from a hydrophilic polymer, 
such as starch or gelatin. In preparing the hard capsules, the liquid 
formulation will be poured into the shell component and then the capsule 
will be sealed by fitting the cap component over the shell component. The 
seal between the two components may be secured, thereby preventing leakage 
of the enclosed formulation from the capsule, by using a sealant as 
described in EP 116744, the disclosure of which is herein incorporated by 
reference. To avoid degradation in the stomach, capsules comprising the 
subject formulations may be coated with an enteric coating which inhibits 
degradation of the capsule in the acidic environment of the stomach. A 
variety of enteric coatings are known in the art. See for example, U.S. 
Pat. No. 5,206,219, the disclosure of which is herein incorporated by 
reference. 
The subject formulations find use in immunosuppressive therapy. 
Immunosuppressive therapy is indicated in a wide variety of diseases, 
including idiopathic nephrotic syndrome, type I insulin-dependent 
diabetes, Behcet's syndrome, active Crohn's disease, aplastic anemia, 
severe corticosteroid-dependent asthma, psoriasis and other diseases where 
the immune system may play a pathogenic role. Of particular interest is 
the use of the subject formulations in transplant situations, including 
both allogeneic and xenogeneic organ, tissue or cell transplantation, 
where immunosuppression is desired to ensure maintained viability of the 
transplanted organ or tissue or cell following transplantation, i.e. to 
prevent graft rejection or prevent graft vs. host disease, e.g. following 
bone marrow transplantation. 
In using the subject formulations to provide immunosuppressive therapy to a 
host, an effective amount of cyclosporin will be orally administered to 
achieve the desired level of immunosuppression in the host, depending on 
the particular condition to be treated. With transplantation, usually an 
initial dosage of cyclosporin will be administered prior to operation. 
Following transplantation of the donor organ to the host, the cyclosporin 
will be administered repeatedly, i.e. chronically, to the host to maintain 
immunosuppression. The initial dosage will be administered 4 to 12 hours 
prior to transplantation and may range from 10 to 18 mg/kg host, usually 
10 to 15 mg/kg host. Following the operation, the initial dosage will 
usually be continued on a daily basis for a period of 1 to 3 weeks, 
usually 1 to 2 weeks. The dosage may then be tapered to a maintenance 
dosage of 3 to 10 mg/kg per day, usually 3 to 6 mg/kg per day. The rate at 
which the dosage is tapered to the maintenance level may range from 3 to 
8% per week and will usually be about 5%/ week. The dosage will typically 
be adjusted based on trough blood levels to maintain a concentration of 
150 to 250 .mu.g/ml, as measured by HPLC, RIA, ELISA or Tdx assay. The 
subject formulations may be administered in conjunction with additional 
agents, where adjunct therapy is recommended and is known in the art. For 
example, the subject formulations may be administered in conjunction with 
adrenal corticosteroids and azathioprine. 
Administration of the subject formulations in conjunction with 
transplantation of a donor organ to a host will result in a prolongation 
of the viability of the donor organ in the host as a result of suppression 
of the host's immune response to the presence of the donor organ. By 
"prolongation of viability" is meant that the donor organ remains viable 
in the host for a longer period of time than it would have had 
immunosuppressive therapy not been employed in conjunction with the 
transplantation. Thus, prolongation of viability includes maintenance of 
viability for an indefinite period of time. A donor organ is considered 
viable as long as it maintains functionality in the host environment. 
The following examples are offered by way of illustration and not by way of 
limitation. 
EXPERIMENTAL 
The following oral Cyclosporin A formulations were prepared. In each case, 
100 mg CsA, the indicated amount of surfactant, and the indicated amount 
of ethanol or propylene glycol were added to a 1.0 ml volumetric flask, 
and the final volume of 1.0 ml was achieved by addition of a suitable 
volume of fatty acid ester. 
______________________________________ 
Formulation 
Composition 
______________________________________ 
19 EtOH 0.1 ml (10%) 
Tween 80 300 mg (0.278 ml) 
IM q.s. to 1.0 ml 
(0.622 ml) (531 mg) 
20 EtOH 0.05 ml (5%) 
Brij 30 350 mg (0.368 ml) 
IM q.s. to 1.0 ml 
&lt;(0.582 ml)(496 mg) 
21 PG 0.05 ml (5%) 
Brij 30 350 mg (0.368 ml) 
IM q.s. to 1.0 ml 
&lt;(0.582 ml)(496 mg) 
22 EtOH 0.1 ml (10%) 
Tween 80 300 mg (0.278 ml) 
EO q.s. to 1.0 ml 
&lt;(0.622 ml)(541 mg) 
23 EtOH 0.05 ml (5%) 
Brij 30 350 mg (0.368 ml) 
EO q.s. to 1.0 ml 
&lt;(0.582 ml) (506 mg) 
24 PG 0.05 ml (5%) 
Brij 30 350 mg (0.368 ml) 
EO q.s. to 1.0 ml 
&lt;(0.582 ml) (506 mg) 
33 EtOH 0.1 ml (10%) 
Brij 30 150 mg (0.158 ml) 
IM q.s. to 1.0 ml 
&lt;(0.742 ml)(633 mg) 
34 EtOH 0.1 ml (10%) 
Brij 30 150 mg (0.158 ml) 
EO q.s. to 1.0 ml 
&lt;(0.742 ml)(646 mg) 
35 EtOH 0.1 ml (10%) 
Tween 80 500 mg (0.463 ml) 
PG q.s. to 1.0 ml 
&lt;(0.437 ml)(453 mg) 
36 EtOH 0.1 ml (10%) 
Tween 80 300 mg (0.278 ml) 
PG 100 mg (0.097 ml) 
EO q.s. to 1.0 ml 
&lt;(0.525 ml)(465 mg) 
37 EtOH 0.1 ml (10%) 
Tween 80 300 mg (0.278 ml) 
PEG 400 100 mg (0.088 ml) 
EO q.s. to 1.0 ml 
&lt;(0.534 ml)(464 mg) 
38 EtOH 0.1 ml (10%) 
Brij 30 300 mg (0.316 ml) 
PG 100 mg (0.097 ml) 
EO q.s. to 1.0 ml 
&lt;(0.487 ml)(424 mg) 
39 EtOH 0.1 ml (10%) 
Brij 30 300 mg (0.316 ml) 
PG 200 mg (0.193 ml) 
EO q.s. to 1.0 ml 
&lt;(0.391 ml)(340 mg) 
40 PG 300 mg (290 ml) 
Brij 30 300 mg (0.316 ml) 
EO q.s. to 1.0 ml 
&lt;(0.394 ml)(343 mg) 
41 EtOH 0.05 ml (5%) 
Brij 30 150 mg (0.158 ml) 
Tween 80 100 mg (0.093 ml) 
EO q.s. to 1.0. ml 
&lt;(0.649 ml) (565 mg) 
42 PG 0.05 ml (5%) 
Brij 30 150 mg (0.158 ml) 
Tween 80 100 mg (0.093 ml) 
EO g.s. to 1.0. ml 
&lt;(0.649 ml) (565 mg) 
______________________________________ 
PG = Propylene Glycol 
EtOH = ethanol 
Brij 30 = polyoxyethylene (4) lauryl ether 
Tween 80 = polyoxyethylene (20) mono sorbitan monooleate 
IM = isopropyl myristate 
EO = ethyl oleate 
The pharmacokinetic properties of cyclosporin in each of the above 
formulations was studied as follows. For each of the above formulations, 
the following pharmacokinetic parameters were determined: (a) the peak 
blood concentration of cyclosporin (C.sub.max); (b) time required to 
attain C.sub.max (T.sub.max) ; and the area under the curve time-profile 
(AUC). For each of the above formulations, as well as a SANDIMMUNE.RTM. 
Oral Solution (SO), CsA-naive Sprague Dawley rats weighing 250-350 gm were 
fed pelletized standard food (Agway.RTM. 3000, Granville Mill, Greensboro, 
N.C.) and water ad libitum. One day prior to the experiment, silicone 
rubber cannulae were inserted into the right jugular and right femoral 
veins under light ether anesthesia. After overnight fast, CsA was 
administered by gavage. 
Following administration, blood samples, 200 .mu.l each, were collected 
from the jugular vein in 0.5 ml polypropylene microfuge tubes containing 
0.3 mg of lyophilized Na EDTA and vortexed immediately for 10 sec. The 
sampling times for animal subjected to oral formulations were 0, 0.5, 1, 
2, 4, 8, 12, 24, 36, 48 and 72 hr after administration. 
CsA, including some of its metabolites, was determined in whole blood by 
fluorescent polarization immunoassay (FPI) (Tdx, Abbot Lab.). Briefly, 150 
.mu.l of the whole blood sample were quantitatively transferred to a 1.5 
ml microfuge tube. Cells were lysed and dissolved with 50 .mu.l of a 
surfactant-containing solubilizing reagent. Proteins are then precipitated 
out with 300 .mu.l of acetonitrile. After centrifugation, the supernatant 
was subjected to the FPI assay in a TDx Autoanalyzer following the 
procedure recommended by Abbott Diagnostics. Since the TDx assay was 
originally developed for human blood, some of the recommended procedures 
were modified as follows. A series of standard solutions of known CsA 
concentration were prepared by adding a known amount of CsA to rat blood 
treated with EDTA. When the CsA concentration in a sample was expected to 
be greater than 1.0 .mu.g/ml, the blood sample was diluted 10-fold in a 
0.1 M-phosphate buffer at pH 7.0. For diluted samples, another calibration 
curve was made using a series of standard solutions containing known 
amounts of CsA, which is volume-wise 10% in rat blood and 90% phosphate 
buffer. 
Descriptive pharmacokinetic parameters were obtained from non-compartmental 
analyses. The peak concentration (C.sub.max) and the time at which the 
peak concentration occurred (T.sub.max) were estimated by inspection of 
the raw concentration-time profile for each rat. The area under the 
concentration-time profile (AUC) from time 0 through the last data point 
(AUC.sub.0-t) was calculated according to the linear trapezoidal 
procedure. The residual area under the tail of the concentration-time 
profile (AUC.sub.t-.varies.) was estimated as the ratio of the final 
observed concentration (C*) to the first-order rate constant associated 
with the terminal elimination phase of the concentration-time profile 
(.lambda..sub.z). The rate contact .lambda..sub.z was determined by 
log-linear regression of the concentration-time data in the apparent 
terminal log-linear phase of the concentration-time profile (i.e., the 
final 3 to 5 data points, depending on the profile under analysis). The 
total AUC (AUC.sub.t-.varies.) was taken as the sum of AUC.sub.0-t and 
AUC.sub.t-.varies.. 
The results for each formulation were compared with the results obtained 
for SO, and are provided in FIGS. 1-3. 
From the above results and discussion, it is evident that novel cyclosporin 
formulations having high bioavailability are provided. The subject 
formulations are capable of comprising high concentrations of cyclosporin. 
The subject formulations are amenable to delivery in capsule form, 
including hard capsule form, providing for ease of storage and handling. 
All publications and patent applications cited in this specification are 
herein incorporated by reference as if each individual publication or 
patent application were specifically and individually indicated to be 
incorporated by reference. 
Although the foregoing invention has been described in some detail by way 
of illustration and example for purposes of clarity of understanding, it 
will be readily apparent to those of ordinary skill in the art in light of 
the teachings of this invention that certain changes and modifications may 
be made thereto without departing from the spirit or scope of the appended 
claims.