Note: Descriptions are shown in the official language in which they were submitted.
1 341 39 6
NBiI CICLOSPORIN CRYSTAL FOR!!, PROCESS FOR ITS PRODIJCTTOI~1,
PHARMACEUTICAL COMPOSITIONS CONTAINING IT AI~iD ITS USE.
The present invention relates to novel pharmaceutical compositions
comprising Ciclosporin as active ingredient.
Ciclosporin, or cyclosporin A, is a known fungal metabolite having
valuable pharmaceutical utility. Ciclosporin is a cyclic poly-N-
methylated endecapeptide of formula
MeBmt-aAbu-Sar-MeLeu-Val-MeLeu-Ala-(D)Ala-MeLeu-MeLeu-MeV
1 2 3 4 5 6 7 8 9 10 11
in which -MeBmt- represents an N-methyl-(4R)-4-but-2E-en-1-yl-4-
methyl-(L)threonyl residue.
Ciclosporin as well as methods for its production are, for example,
described in US patent no. 4,117,11$. The primary area of clinical
investigation for ciclosporin has been as an immunosuppressive agent,
in particular in relation to its application in treating recipients of
organ transplants, e.g. heart, lung, combined heart-lung, liver, kid-
ney, pancreatic, bone-marrow, skin arid corneal transplants and, in
particular, allogenic organ transplants. In this field Ciclosporin has
achieved a remarkable success and reputation and is now widely emplo-
yed in clinic and commercially available under the Registered Trade
Mark SANDIMMUNR or SANDIMMUNER, for example in the form of an infusion
concentrate, of an oral solution and of a gelatin encapsulated
solution, the latter two variants being described and claimed for
example in US patent no. 4,388,307.
1341396
_ 2 -
At the same time, investigation of the applicability of Ciclosporin to
various autoimmune diseases and to inflammatory conditions, in parti-
cular inflammatory conditions with an aetiology including an auto-
immune component such as arthritis (for example rheumatoid arthritis,
arthritis chronica progrediente and arthritis deformans) and rheumatic
diseases, has been intensive and reports and results in vitro, in
animal models and in clinical trials are wide-spread in the litera-
ture. Specific auto-immune diseases for which Ciclosporin therapy has
been proposed or applied include, autoimmune hematological disorder
(including e.g. hemolytic anaemia, aplastic anaemia, pure red cell
anaemia and idiopathic thrombocytopaenia), systemic lupus erythema-
tosus, polychondritis, sclerodoma, ilegener granulamatosis, dermatomy-
ositis, chronic active hepatitis, myasthenia gravis, psoriasis,
Steven-Johnson syndrome, idiopathic sprue, autoimmune inflammatory
bowel disease (including e.g. ulcerative colitis and Crohn~s disease),
pemphigus, endocrine opthalmopathy, Graves disease, sarcoidosis,
multiple sclerosis, primary billiary cirrhosis, primary juvenile
diabetes (diabetes mellitus type I), Behcet's disease, uveitis
(anterior and posterior), vernal conjunctivitis, keratoconjunctivits
sicca, interstitial lung fibrosis, psoriatic arthritis and
glomerulonephritis (with and without nephrotic syndrome, e.g.
including idiopathic nephrotic syndrome or minimal change
nephropathy).
Further areas of investigation have included potential applicability
as an anti-parasitic, in particular anti-protoaoal agent, with
possible uses suggested including treatment of malaria,
coccidiomycosis and schistosomiasis, as well as use in reversing tumor
resistance to chemotherapeutic, e.g. cytostatic, therapy and in
promoting hair growth, e.g. for the treatment of alopecia.
In the solid stake, Ciclosporin exists in both amorphous and crystal-
line form. In the crystalline state, both a tetragonal and an
orthorhombic modification are known.
The tetragonal modification (P41), also referred to herein for con-
-3- ~ 3 41 3 9 6
venience as "Cy-A/X-I", is described together with processes
for its production, e.g. in Austrian patent specification No.
353,961. Cy-A/X-I has a lance a=b= 13.8 A c= 41.2 A, volume
per asymmetric unit = 1974 A~, a melting point at ca.
140-150 °C and comprises ca. 2 molecules HZO per Ciclosporin
molecule.
A first orthorhombic modification (P2~2~2~) is obtainable by
recrystallisation from di-isopropyl ether as discussed in an
article entitled "Total synthesis - change in molecular
structure - biological effect: Ciclosporin as example" by
R. Wenger, in "Sandorama" 1984/III. This particular
modification comprises a Ciclosporin/di-isopropyl ether (ca.
1:2) solvate, with a crystal lattice of a = 12.5 A, b = 22.9
A, c = 28.4 fir, vol per asymmetric unit = 2027 A3, and a melting
point at ca. 150 °C. Orthorhombic Ciclosporin solvate
crystals are obtainable by recrystallisation of supersaturated
Ciclosporin solutions in an appropriate solvent medium over
prolonged periods of time. Thus the orthorhombic
Ciclosporin/di-isopropyl ether solvate modification prepared
by Wenger is obtained by addition of a concentrated solution
of Ciclosporin in methylene chloride (in which Ciclosporin is
rather readily soluble) to di-isopropyl ether (in which
Ciclosporin is relatively less soluble) and evaporation of the
methylene chloride. Crystallisation from the remaining di-
isopropyl ether is then allowed to proceed over a period of
ca. 7 days at ambient temperature. Orthorhombic crystal forms
comprising such Ciclosporin solvates are also referred to, for
convenience, herein generically as "CY-A/X-II".
A further orthorhombic (P2~2~2~) modification has now been
identifed, characterised by the absence or substantial absence
of any solvent component, i.e. consisting substantially of, or
essentially of, free Ciclosporin.
~",.,.
~ 341396
-3a-
Brief Description of the Drawings
In the drawings which illustrate preferred embodiments of the
invention:
Figs 1, 2 and 3 are plots of skin thickness v. test time for
three different test materials, and
Fig 4 shows X-ray powder diffractions for three materials.
The dimensions of the crystal lattice for solvent free or non-
solvate, orthorhombic Ciclosporin modification are a = 12.7 A,
b = 15.7 A, c = 36.3 A, vol. per asymmetric unit = 1804 A3.
The melting point is of the order of from about 180 to about
190 °C up to about 195 °C, e.g.
.....
13~~ 39s
_ t, _
depending on degree of purity/source as indicated in Examples 1 and 2
hereinafter, in which specific methods for the obtention of this
modification are described. Ciclosporin in non-solvate (or solvent
free) orthorhombic crystal form is also referred to herein for
convenience as "CY-A/X-III". Further characterising (X-ray powder
diffraction) data for CY-A/X-III are provided in Table III and Fig. IV
hereinafter, together with corresponding data for CY-A/X-.I and
CY-A/X-II.
Despite the very considerable success and reputation which Ciclosporin
has achieved and despite the major contribution to medical science, in
particular to the art of organ transplant, which it has already made,
its use in practice is still not ideal or problem free. A particular
area of difficulty which has been encountered is in the provision of
galenic forms permitting convenient or wholly satisfactory administra-
tion, or best suited for the treatment of specific diseases or condi-
tions for which Ciclosporin has already been identified as a potential
drug of choice.
These difficulties are in part attributable to the particular physical
characteristics of Ciclosporin, for example its relatively limited
solubility or (hitherto experienced) compatibility with available,
pharmaceutically applicable carrier media. Such difficulties are
compounded by the very nature of certain of the disease conditions for
which Ciclosporin represents potential first line therapy as well as
the occurrence of undesirable side effects which Ciclosgorin may
induce when administered orally. For example Ciclosporin has been
identified as effective in the treatment of psoriasis following oral
administration. Psoriasis is a wide-spread condition causing
considerable patient suffering and for which there is at present no
generally accepted, effective therapy. Sufferers from psoriasis
clearly represent a patient population at need. While available
evidence would suggest that Ciclosporin might meet this need it is
improbable that oral Ciclosporin therapy could be ,justified as a con-
ventional means of psoriasis treatment. Similar considerations apply
to the treatment of e.g. eye diseases such as posterior uveitis and
...-. ,
v..~
- 1341396
_5_
keratoconjunctivitis sicca.
Various systems have been proposed for the administration of
Ciclosporin, including galenic forms intended for topical or dermal
application or for ophthalmic application, i.e. topical application to
the eye, e.g. for possible use in the treatment of psoriasis, of other
dermatological diseases and conditions, for example atopic dermatosis
and alopecia, or for the treatment of uveitis. Such systems have
however hitherto not brought any notable benefit, or have not proven
wholly satisfactory.
Thus variously reported attempts to treat psoriatic subjects by the
application of the known oral solution directly at the superficial
site of psoriatic lesion have met with minimal or no success and
success in relation to subjects suffering from atopic dermatosis has
been strictly limited. Similarly, while topical application to the eye
has been found to be widely effective as a means of preventing e.g.
corneal transplant rejection, or of treating disease in the anterior
segment of the eye, no topical formulation has hitherto been reported
which might permit ready, safe or effective treatment of diseases or
conditions as they affect sites deeper within the eye, far example for
the general treatment of uveitis, including posterior uveitis.
Comparable difficulties have been encountered in relation to the
development of other ciclosporin drug delivery systems, for example
injectible forms, e.g. suitable for intra-articular injection for the
local treatment of arthritic diseases, e.g. as hereinbefore set forth,
or for intra-lesional injection, e.g. for the treatment of severe
psoriatic lesion, or alternative oral delivery systems, e.g. having
modified bioavailability or drug-release characteristics.
Hitherto attempts to formulate Ciclosporin have made use of the
compound in solution or, less commanly, in amorphous or Cy-A/X-I form,
the latter being, for example, the crystal form identified e.g. for
drug registration purposes. And despite the very considerable efforts
expended in seeking new or improved alternatives, the oral solution
M,~. 1 3 41 3 9 6
-6-
and infusion concentrate already referred to remain the only widely
applied delivery systems for Ciclosporin.
In accordance with the present invention, it has now surprisingly been
found that orthorhombic crystalline forms of ciclosporin, for example
Cy-A/X-II and, in particular, Cy-A/X-III, are of especial advantage
and benefit in the preparation of ciclosporin galenic formulations of
various and diverse kind.
In particular it has been found that such crystal modifications may be
used to prepare galenic formulations comprising Ciclosporin in stable,
fine particulate form and/or having enhanced stability or release
characteristics. By this finding the way is opened to the production
of novel and improved Ciclosporin drug delivery systems, e.g.
possessing sustained or delayed Ciclosporin delivery characteristics
and/or adapted to or suitable for the treatment of diseases or
conditions hitherto refractory to Ciclosporin therapy or for which
conventional Ciclosporin therapy has been inherently less suited.
More especially, it has been found that by use of such crystal
modifications, e.g. in fine particulate form as aforesaid, galenic
forms may be obtained suitable for topical or dermal application or
for topical ophthalmic application, for the treatment of diseases or
conditions affecting the skin or eye, e.g. as hereinbefore set forth,
in particular for the treatment of psoriasis, atopic dermatitis,
contact dermatitis, occular symptoms of Behchet's disease, uveitis and
keratoconjunctivitis sicca. Use of such crystal modifications also
enables the preparation of injectible forms for Ciclosporin having
sustained or delayed release characteristics and hence of particular
utility, e.g. in the treatment of diseases or conditions for which
required frequency of administration may otherwise impede utility, for
example for the treatment of arthritic or similar disease of the joint
by intra-articular injection or for the treatment of psoriasis or
similar diseaseJof the skin by intra-lesional injection.
For such purposes orthorhombic crystal forms, in particular
CY-A/X-III, have been found to be inherently much better suited than
1 341 39 6
_,_
other available forms, in particular amorphous Ciclosporin or
CY-A/X-I, e.g. in terms of physico-chemical properties, especially
stability, release characteristics, i.e. release of Ciclosporin from
the crystal lattice, and susceptibility to the necessary procedures of
galenic formulation. CY-A/X-III is new and the preferred orthorhombic
crystal form for galenic and/or therapeutic use in accordance with the
invention. In a first embodiment the present invention accordingly
provides:
A CY-A/X-III, 1.e. Ciclosporin in non-solvate orthorhombic crystal
form;
in particular:
A1 CY-A/X-III having a crystal lattice or other physical
characteristic substantially as hereinbefore defined, and/or
Az CY-A/X-III having X-ray powder diffraction characteristics
substantially as herein set forth in Table III or as represented
in Fig. IV.
The present invention also provides:
B A process for the production of CY-A/X-III which process comprises
crystallising Ciclosporin from a CY-A/X-III bearing medium and
recovering CY-A/X-III thus obtained.
Suitable CY-A/X-III bearing media far use in the above process are, in
particular, solvent media for Ciclosporin comprising higher molecular
weight solvent components, e.g. having a molecular weight >200.
Especially suitable are media comprising higher molecular weight
ethers, in particular polymeric ethers, for example polyalkyl ethers,
e.g. polyethylene-, and polypropylene-glycols. Examples of polymeric
ethers which have been found to be appropriate are polyethylene and
polypropylene glycols, e.g. having a molecular weight of >200;
glycerin-polyethyleneglycol esters, e.g. glycerin-polyethylene-
glycolricinoleates and glycerin-polyethyleneglycoloxystearates as, for
1341396
_ 8 _
example, available under the Trade Marks "Cremophor EL" and "Cremophor
RH" (c. f. Fiedler, "Lexikon der Hilfstoffe" 2nd edition, Vol. 1, p.p.
257 and 258); polyoxyethylene-sorbitan esters, for example as
available under the Trade Mark "Tween" (c. f. Fiedler, 2, p.p.
972-975); polyoxyethylene esters, e.g. polyoxyethylene-stearinic acid
esters, for example as available under the Trade Mark "Myrj" (c. f.
Fiedler, 2, p.p. 636); and trans-esterification products of natural
oil triglycerides and polyalkylene polyols, including the
trans-esterification products of maize oil, kernel oil, almond oil,
ground nut oil, olive oil, palm oil and mixtures thereof with
polyethylene-glycols, in particular polyethylene-glycols having a
molecular weight of from Z00 to 800, for example as available under
the Registered Trade Mark "Labrafil" (c. f. Fiedler, p. 539).
Especially suitable are media comprising polyethylene-glycols having a
molecular weight of ca. 200 to ca. 600, in particular of ca. 300 to
ca. 400.
As will be appreciated, the CY-A/X-III bearing medium may include one
or more components in addition to those recited above, for example
lower molecular weight alcohols and/or water, as a means of modifying
solvent/crystallisation characterisitics. A particularly suitable
CY-A/X-III bearing medium has been found to be one comprising ca. 0.5
parts by volume ethanol, ca. 8.0 parts by volume polyethylene-glycol
(300) and ca. 1.0 parts by volume H20.
The process of the invention comprises first dissolving Ciclosporin,
e.g. in amorphous or tetragonal form in the selected CY-A/X-III
bearing medium at elevated temperature. The dissolution temperature
will of course vary depending on the particular medium chosen but will
generally be >40°C more especially >50°C. In the case of
polyethylene-glycol based media the dissolution temperature will
generally be >70°C, e.g ca. 75-130°C. Suitably the obtained
solution
will comprise >5%, preferably >10%, e.g. up to about 40-64%,
preferably up to a maximum of 20% by weight Ciclosporin based on the
total weight of the solution. Crystallisation of Ciclosporin is then
allowed to proceed with cooling, suitably over a relatively prolonged
1 341396
- 9 -
period of time, e.g. of the order of 10-35 minutes or mare depending
on desired crystal growth.
If desired nucleation procedures may be commenced, e.g. by sonication
or seeding. Where ethanol/polyethylene-glycol/Hz0 crystal bearing
media, e.g. as described above, are employed it has, for example, been
found that nucleation can be readily initiated at te~aperatures of ca.
60-90°C, e.g. ca. 65-85°C, by application of sonic vibration at
ca.
20,000 c.p.c. As indicated in example 2 hereinafter, by continuing
sonication in this manner throughout the course of crystallisation,
micro crystals may be obtained of very fine and relatively constant
particle size.
The rate of crystal growth and the size of crystals obtained will of
course, depend on the particular medium chosen and the conditions
under which crystallisation is performed, for example on whether or
not sonication is applied and at what frequency. For use in accordance
with the present invention, e.g. for the preparation of galenic
formulations as hereinafter described, CX-A/X-III will preferably be
employed in fine particulate form, e.g. having an average particle
size of <200uM, e.g. of from 0.5 to 200uM. Where initially obtained
crystals are of relatively large size, particulate preparations having
the desired size characteristics can, of course, be obtained by
conventional comminution procedures, e.g. by grinding, milling,
pulverising, micronising or otherwise fragmenting to reduce size.
While particulate preparations obtained in this way may be entirely
adequate for some purposes, for example in the preparation of topical
compositions for application to the skin, use of grinding or milling
or equivalent techniqes will in many cases not be preferred. Reasons
for this are that the products of such procedures tend to exhibit
relatively wide particle size distribution and to have a relatively
coarse or abrasive structure as a consequence of crystal fracture. In
addition comminution will tend to destroy physical characteristics at
the natural crystal surface and is in any event difficult or costly to
perform while maintaining required conditions of sterility, e.g. in
the manufacture of galenic formulations for intra-articular
..y. 1 3 4 1 3 9 fi
- to -
administration. For such reasons it is commonly preferred to carry out
the method of the invention in such a manner as to obtain crystals,
e.g. microcrystals, of dimensions within the desired end particle size
range. Thus for use in the manufacture of e.g. compositions for
intra-articular or other parenteral application use of CY-A/X-III
particle preparations in non-comminuted microcrystalline state (i.e.
CY-A/X-III microcrystals in native or non-fractured condition, e.g.
which have not been subjected to grinding, milling, micronization or
other reductive procedures) will be preferred.
In order to obtain CY-A/X-III microcrystals which can be used directly
in non-comminuted state in the above described manner, the process of
the invention will suitably be carried out with agitation, for
example by stirring, e.g. at 200-340 r.p.m. rising to e.g. 700 r.p.m.
during crystallisation, and, especially, by application of ultra-sonic
vibration, e.g. within the range of 10,000-30,000 cycles per sec,
suitably of the order of about 20,000 cycles per sec.
By variation of the CY-A/X-III bearing medium and adjustment of the
rate of cooling and degree of agitation during the recrystallisation
procedures in accordance with techniques known in the art and as
hereinafter exemplified, CY-A/X-III microcrystals may be produced of
varying average particle size, e.g. as set forth above or as
hereinafter described for use in relation to individual types of
galenic formulation in accordance with the invention.
As previously indicated crystallisation in accordance with the process
of the invention will suitably be performed under sterile conditions.
Purification of the obtained CY-A/X-III may be carried out in
conventional manner, e.g. by rinsing with poly ethylene glycol (300)
water e.g. in a ratio of 3-4:1 p.p.w./ washing with warm water, as
described in the examples. The obtained CY-A/X-III will suitably be
recovered free or substantially free of Ciclosporin in any other form,
preferably in pure or substantially pure form. The rinsing step above
assists prevention of the formation of amorphous Ciclosporin.
1341396
- 11 -
In accordance with the foregoing the present invention additionally
provides: Ciclosporin producible by any of the above described
procedures as well as:
A3 CY-A/X-III according to any one of A to Az above, free or
substantially free of Ciclosporin in any other form, e.g. free or
substantially free of Ciclosporin in amorphous form, of CY-A/X-I
or of CY-A/X-II;
A4 CY-A/X-III according to any one of A to A3 above in pure or
substantially pure form;
A5 CY-A/X-III according to any one of A to A° above in fine
particulate form, e.g. having particle size characteristics as
hereinbefore set forth or as hereinafter described, e,g. in
relation to galenic forms comprising CY-A/X-IIT;
A6 CY-A/X-III according to any one of A to AS above consisting
predominantly, or consisting entirely or substantially entirely,
of non-comminuted crystal material, e.g. CY-A/X-III according to
A5 above wherein the component particles are comprised
predominantly, or comprised entirely or substantially entirely, of
non-comminuted microcrystals; and
A~ CY-A/X-III according to any one of A to A6 above in sterile or
substantially sterile condition, e.g. in a condition suitable for
pharmaceutical use, in particular suitable for parenteral
application, e.g. for administration by parenteral injection, e.g.
intra-articular injection.
In addition the present invention also provides:
C A pharmaceutical composition comprising
a) Ciclosporin in orthorhombic crystal form as active ingredient,
together with
b) a pharmaceutically acceptable diluent or carrier therefor.
..~ ~ 3~~3~s
- 12 -
Ciclosporin may be present in the compositions of the invention in any
appropriate orthorhombic crystal form, e.g. as CY-A/X-II or
CY-A/X-III. Use of CY-A/X-II will however generally be less preferred
having regard to its physico-chemical properties which are less well
suited for use both in terms of tolerability and useability for the
manufacture of effective Ciclosporin-based pharmaceutical products.
Most preferably Ciclosporin will be present in the compositions of the
invention as CY-A/X-III, e.g. according to any one of A to A~ above.
Though Ciclosporin is present in the compositions of the invention in
solid (crystalline) form, the compositions themselves may be of any
appropriate constitution. Compositions in accordance with the inven-
tion thus include solid forms, such as capsules, tablets, dragies,
powders and granulates, semi-solid forms such as ointments, gels,
creams and pastes, as well as liquid forms, e.g. comprising the
defined Ciclosporin component in fine particulate form (for example
micronised or microparticuiate form) suspended or dispersed in a
liquid pharmaceutical diluent or carrier, in which the chosen crystal
form may be retained.
Compositions in accordance with the invention include both systems
comprising a single Ciclosporin-containing phase or compartment, for
example simple creams, gels, ointments etc... as aforesaid or simple
one compartment capsules or ampoules, for example hard or soft gelatin
capsules or ampoules for injection, as well as systems comprising a
plurality of phases or compartments. Thus the invention is to be
understood as embracing, e.g. compositions comprising a Ciclosporin-
containing phase or compartment together with one or more
non-Ciclosporin-containing phases or compartments, e.g. coating,
carrier, retard or placebo phases, or phases or compartments
comprising other drug substances or adjuvant materials. The invention
is thus to be understood as embracing, for example: coated tablets
having a core comprising Ciclosporin in orthorhombic crystal form,
surrounded e.g. by other non-Ciclosporin-containing coating phases or
the like; mantle tablets comprising a core or mantle comprising
Ciclosporin in orthorhombic form and provided with a
-13- 1341396
non-Ciclosporin-containing mantle or core; mixed granulates comprising a first
granulate
phase comprising Ciclosporin in orthorhombic crystal form and a second non-
Ciclosporin-
containing granulate phase; and split capsules having two or more
compartments, e.g.
divided by an internal wall, one of which contains Ciclosporin in orthorhombic
form and
one of which does not. Other alternatives and variants will be apparent to
those skilled in
the art.
Preferably Ciclosporin will be present on the compositions of the invention in
a major
proportion in orthorhombic crystal form, e.g. as CY-A!X-III. It is
particularly preferred
that the Ciclosporin be present entirely or substantially entirely in said
orthorhombic
crystal form.
The compositions of the invention will suitably comprise at least one
phase or compartment comprising Ciclosporin in orthorhombic crystal
form, e.g. CY-A/X-III, throughout which said crystal form is
distributed or distributable uniformly. Such systems include for
example homogeneous creams, ointments, gels and the like as well as
liquid systems in which the contained crystalline Ciclosporin is or
can be readily distributed homogenously, for example by hand
agitation.
Compositions in accordance with the invention may include: forms
suitable for topical administration e.g. dermal or topical ophthalmic
administration; for parentaral administration, e.g. by infusion or
injection, including sub-cutaneous or intramuscular injection and, in
particular, intra-lesional or intra-articular injection; as well as
forms for enteral administration, for example suppositories,
pessaries and the like and oral dosage forms. Enteral forms, for
example oral dosage forms will however generally be less preferred.
Preferably orthorhombic crystalline Ciclosporin, e.g. CY-A/X-III, will
be present in the compositions of the invention in fine particulate
form, e.g. micronised, microparticulate or non-comminuted
microcrystalline form as hereinbefore described. Appropriate particle
size will of course vary depending on the particular nature of the
composition, its intended use and mode of application and the effect
desired. In general however, average particle size will suitably be of
~ 341 396
-m-
the order of from 0.5-200uM~ Particular ranges, e.g. in relation to
the preparation of oral, topical or in~ectible forms are further
discussed hereinafter.
TOPICAL FORMS e.g' for dermal or topical ophthalmic administration,
e.g. for the treatment of psoriasis or atopic dermatosis or of
diseases or conditions of the eke requiring immunosuE~rressive therapy,
for exam 1e uveftis, con unctivitis includin keratocc~r~ unctivits
sicca or vernal keratoconjunctivitis, or corneal transplant.
Gels, creams, ointments and the like for topical or dermal admini-
stration can be prepared in accordance with any of the techniques
known and generally employed in the art, for example by compounding
Ciclosporin in orthorhombic crystal form with an appropriate flowable
base material, e.g. aqueous gel, for example aqueous cellulose-ether
based gel, with the addition of appropriate tensides, thickeners etc.
including e.g. aerosil.
The Ciclosporin component is preferably present in fine particulate
form, e.g. having an average particle size of from 0.5-200uM.
Preferably the average particle size is <60-80uM, more preferably
<40uM~ yet more preferably <30uM, most preferably <20uM.
Advantageously the particles will have as narrow a particle size
distribution as possible. Preferably substantially all particles
present will be less than 60-80uM.
Gompositions of the invention for topical ophthalmic use, i.e.
application to the eye, may include, for example suitably thickened
suspensions or dispersions presented e.g. in the form of viscous
eye-drop preparations or creams or gels for occular application. In
the case of compositions for administration to the eye, the average
particle size for the crystalline Ciclosporin material present will
suitably be <25uM. More preferably substantially all particles present
will be less than 25uM. Average particle size for such eye drop
preparations will suitably be in the range of from about 0.1, or
preferably from about 0.5, to about 20uM or, preferably, to about lOuM.
..-.
1 341 396
- 15 -
The hydrophilic/lipophilic content of the carrier system for use in
forms for dermal application or for use in preparations for application
to the eye may vary, e.g. depending on the particular surface to which
the composition is to be applied, e.g. depending on the characteristics
of the particular skin area to be treated.
Compositions for topical, in particular dermal, application suitably
comprise a carrier medium in which the active ingredient (Ciclosporin
component) is only slightly soluble. Suitably the solubility of the
Ciclosporin component (e. g. CY-A/X-III) in such compositions is <10X,
preferably <1.0X by weight. More preferably solubility is <0.1X, most
preferably <0.001% by weight.
Suitable carrier media include hydrophilic, water miscible carriers as
well as hydrophobic carriers.
Hydrophilic, water miscible carrier components are suitably of
relatively low volatility. Examples of appropriate carrier components
belonging to this class include liquid polyalkylene glycols, propylene
glycol, glycerol and ethylene glycol. Suitable polyalkylene glycols
include in particular, liquid polyethylene glycols, e.g. having an
average molecular weight up to about 600, e.g. having an average
molecular weight from about 250 to 450, especially ca. 300.
Representative compositions in accordance with the invention, e.g. for
topical application, accordingly comprise:
C1 a) Ciclosporin in orthorhombic, preferably non-solvate
orthorhombic, crystal form, e.g. Cy-A/X-III, e.g. as defined
under any one of A to A' above;
b) A hydrophilic, water miscible carrier, e.g. as described above;
and, optionally
c) Pharmaceutical grade water.
~ 341396
- 16 -
The amount of component (a) present will suitably be of the order of
from about 0.1 to about 30.0%, especially about 0.5 or 1.0 to 20%,
e.g. ca. 1.0, 2.0, 5.0 or 10.0% based on the total weight of the
composition. Components (b) and (c) will suitably be present in a
ratio of from 1:0 to 1:20 p.p.w. More suitably components (b) and (c)
will be present in a ratio of from 1:0.5 to 1:5, e.g. from 1:1 to 1:3,
most suitably 1:1.5 to 1;2.5 p.p.w.
Such compositions will suitably additionally comprise:
d) A surface active agent.
Suitable components (d) include, inter al.
dl Traps-esterification products of natural vegetable oil
triglycerides and polyalkylene polyols.
Such traps-esterification products are known from the art and may
be obtained e.g. in accordance with the general procedures
described in US Patent No. 3 288 824. They include
traps-esterffication products of various natural (e. g.
non-hydrogenated) vegetable oils for example, maize oil, kernel
oil, almond oil, ground nut oil, olive oil and palm oil and
mixtures thereof with polyethylene glycols, in particular
polyethylene glycols having an average molecular weight of from
200 to 800. Preferred are products obtained by
traps-esterification of 2 molar parts of a natural vegetable oil
triglyceride with one molar part of polyethylene glycol (e. g.
having an average molecular weight of from 200 to 800). Various
forms of traps-esterification product of the class defined are
known and commercially available under the Trade Mark Labrafil
(see Fiedler, "Lexikon der Hilfstoffe", 2nd. revised and expanded
edition (1981), Vol. 2, page 539]. Especially useful as components
of the compositions of the invention are the products: Labrafil M
1944 CS, a traps-esterification product of kernel oil and
polyethylene glycol having an acid number of ca. 2, a
saponification number of 145- 175 and an iodine number of 60 - 90;
1341396
- 17 -
and Labrafil M 2130 CS, a trans-esterification product of a Clz-
to C18- glyceride and polyethylene glycol having a melting point
of ca. 35 - 40°C., an acid number of <2, a saponification value of
ca. 185 - 200 and an iodine number of <3.
d2 Reaction products of a natural or hydrogenated castor oil and
ethylene oxide. Such products may be obtained in known manner,
e.g. by reaction of a natural or hydrogenated castor oil with
ethylene oxide, e.g. in a molar ratio of from about 1:35 to about
1:60, with optional removal of the polyethyleneglycol component
from the product, e.g. in accordance with the methods disclosed in
German Auslegeschriften 1,182,388 and 1,518,819. Especially
suitable are the various tensides available under the Trade Mark
Cremophor. Particularly suitable are the products Cremophor RH 40
having a saponification number of ca. SO-60, an acid number <1, an
iodine number <1, a water content (Fischer) <2%, an nDSO of ca.
1,453 - 1,4578 and an HLB of ca. 14 - 16; Cremophor Ri~60 having a
saponification number of ca. 40 - 50, an acid number <1, an iodine
number <1, a water content (Fischer) 4.5-5.S%, an npzs of ca.
1,453 - 1,57 and an HLB of ca. 15 - 17; and Cremophore 8L having a
molecular weight (by steam osmometry) of ca. 1630, a
saponification number of ca. 65 - 70, an acid number of ca. 2, an
iodine number ca. 28 - 32 and an nazs of ca. 1,471. Also suitable
for use in this category are the various tensides available under
the Trade Mark Nikkol, e.g. Nikkol HCO-60. The said product Nikkol
HCO-60 is a reaction product of hydrogenated castor oil and
ethylene oxide exhibiting the following characteristics: Acid
number of 0.3; Saponification number of 47.4; Hydroxy value of
42.5; pH (5X) of 4.6; Color APHA = 40; m.p. - 36.0°C; Freezing
point = 32.4°C; Hz0 content (%, KF) = 0.03.
d3 Polyoxyethylene-sorbitan-fatty acid esters or polysorbates e.g. of the
type known and commercially available e.g. under the Trade Marks Tween
and Armotan (c.f. Fiedler, 2, p.p. 745-746 and 972-975) including the
products Tween
'...,
1 341396
- 18 -
20 [polyoxyethylene(20)sorbitanmonolaurate],
40 [polyoxyethylene(20)sorbitanmonopalmitate],
60 [polyoxyethylene(20)sorbitanmonostearate],
65 [polyoxyethylene(20)sorbitantristearate],
80 [polyoxyethylene(20)sorbitanmonooleate],
85 [polyoxyethylene(ZO)sorbitantrioleate],
21 [polyoxyethylene(4)sorbitanmonolaurate],
61 [polyoxyethylene(4)sorbitanmonostearate], and
81 [polyoxyethylene(5)sorbitanmonooleate].
d4 Polyoxyethylene fatty acid esters, for example polyoxyethylene stearic
acid esters of the type known and commercially available under the
Trade Mark Myrj (c. f. Fiedler, 2, p. 636) as well as polyoxyethylene
fatty acid esters known and commercially available under the Trade Mark
Cetiol HE. (c. f. Fiedler, 1, p. 228), and polyoxyethylene fatty alcohol
ethers, for example polyoxyethylene stearylether, oleyl ether, or cetyl
ether, e.g. of the type known under the Trade Mark Brij (c. f. Fiedler,
1, 184-186), e.g. Brij 78 and 96, and Cetomacrogel 1000 (c. f. Fiedler,
1, 229).
d5 Polyoxyethylene-polyoxypropylene co-polymers e.g. of the type known and
commercially available under the Trade Marks Pluronic and Emkalyx (c. f.
Fiedler, 2, p.p. 720-722.
d6 Sorbitan fatty acid esters e.g. of the type known and commercially
available under the Trade Mark Span, for example including sorbitan-
monolauryl, -monopalmityl, -monostearyl, -tristearyl, -monooleyl and
-trioleyl esters - c.f. Fiedler, 2, p.p. 850-851.
d~ Partial glycerides, e.g. products comprising mono- and/or di-glyceride
components, for example mixed mono-1di-/tri-glycerideJfree glycerol
based surfactants as well as, e.g., glycerol monostearates and
glycerol monooleates. Examples of such products include those known and
commercially available under the Trade Marks Imwitor (c.f. Fiedler, l,
491) for example Imwitor 191 and Imwitor 780< and Soft3gen (c. f.
Fiedler, 2, 833) for example Softigen 701.
~ 341 396
- 19 -
de Ionic surfactants or emulsifying agents such as sodium lauryl sulfate
and sodium cetostearyl sulfate.
Components (d) when present are suitably present in an amount of up to
maximally 30X, more preferably maximally 15X, based on the total weight
of the composition. Most suitably components (d) are present in an
amount of from about 0.5 to about 10X, e.g. about 1.0X, based on the
total weight of the composition.
Such compositions will suitably also comprise:
e) A thickening agent.
Suitable components (e) include for example:
e1 Polymethylacrylate resins, e.g. of the type known and commercially
available under the Trade Mark Budispert (c. f. Fiedler, l, 371-372).
e2 Cellulose derivatives including e.g. ethyl-, propyl-, methyl-, hydroxy-
propylmethyl- and carboxymethyl-celluloses.
e3 Polyvinyl resins, e.g. including polyvinylalcohols and
polyvinylpyrrolidones, as well as other polymeric materials including
gelatin, alginates, pectins, gum traganth, gum arabicum, gum xanthan,
and polyacrylic acids such as known and commercially available under
the Trade Mark Carbopol (c. f. Fiedler, 1, 20b-207).
e4 Materials such as silica gel, bentonite, and magnesium-aluminium
silicate.
Components (e) when present are suitably present in an amount of
up to 20%, more preferably up to 10%, based on the total weight of
the composition. Most suitably components (e) are present in an
amount of from about 0.5 to about 15%, e.g. from about 1.0 to
about 3.0% based on the total weight of the composition.
*~. 1 341 39 6
-20-
In addition such compositions also suitably comprise an
anti-microbial agent such as methylparaben, propylpsraben,
benzalconium chloride or benzyl alcohol, e.g. in an amount of from
0.05 to 0.5% or, in the case of benzyl alcohol, up to 2.0%, e.g.
ca. 0.1%, or, in the case of benzyl alcohol, ca. 1.0X by weight
based on the total weight of the composition.
Alternative carrier media include hydrophobic materials, e.g.
which meet the solubility criteria with respect to the active
ingredient hereinbefore described. Further preferred compositions
in accordance with the invention, e.g. for topical application,
accordingly comprise
CZ a) Ciclosporin in orthorhombic, preferably non-solvate
orthorhombic, crystal form, e.g. Cy-A/X-III, E.g. as defined
under anyone of A to A~ above; and
f) A hydrophobic carrier, e.g. as set forth above.
The amount of component (a) present will suitably be as described
above in relation to compositions as defined under C1.
Suitable components (f) include, for example, petroleum
derivatives, including mineral oils, fats and jellys, as for
example petrolatum (mineral fat or petroleum jelly) liquid
petrolatum (white mineral oil or liquid paraffin), or any of the
products known and commercially available under the Trade Mark
Vaseline [c. f. Fiedler loc. cit. 2, 9$6], as well as non-ionic
lanoline based derivatives such as available under the Trade Mark
Amerchol, e.g. Amerchol CAB [Fiedler, 1, 119-120].
Further suitable components (f) include, for example natural or
saturated vegetable oils and fats, e.g. fractionated coconut oils
as known and commercially available under the Trade Mark Miglyol,
e.g. Miglyol 812 (c. f. Fiedler, 2, 616) and peanut and saturated
pea-nut oils, as well as higher fatty or branched chain alcohols
and fatty esters, such as stearic alcohol, cetyl palmitate and
2-octyldodecanol which is known and commercially available under
,...
~ 34~ :~96
- 21 -
the Trade Mark Eutanol G (c. f. Fiedler, 1, 375).
Such compositions rnay also comprise one or more of any of
components (d) and (e) hereinbefore described, as well as one or
more anti-microbial agents, e.g. as hereinbefore described.
In particular such compositions may additionally comprise one or
more components (d), suitably in an amount of up to at most 30X,
more preferably at most 20%, suitably in an amount of from about 1
to about 15X by weight based on the total weight of the
composition.
hydrophobic based compositions in accordance with Ca and
comprising an emulsifier component (d), may also be prepared in
the form of creams or emulsions by the further addition of water.
The obtained creams or emulsions may be either of the oil-in-water
or water-in-oil type. The amount of water present in such
compositions may thus vary from e.g. 5 to 80X based on the total
weight of the composition, suitably from 20 to 709;.
Amounts of components (e)/antimicrobial agent, when incorporated,
will be of the same or similar order to those set forth in
relation to compositions of the invention in accordance with C1
above.
The amount of any topical form applied will, of course, vary
depending on the concentration of the composition, the condition
to be treated and the effect desired, as well , in the case of
dermal application, the area to be covered. In the case of
compositions for dermal application, e.g. in the treatment of
psoriasis or atopic dermatosis, amounts applied will generally be
sufficient to provide a Ciclosporin dosaging of the order of from
about 0.1 to about 5mg/cmZ, e.g. ca. 0.5mg/cm2, with
administration e.g. 2x daily to the desired site. In the case of
ophthalmic preparations, administration may suitably be performed
employing preparations comprising about 0.1 to about 10X, e.g. 1.0
.. 1 341396
- 22 -
or 2.0Y Ciclosporin by weight, applied in drop form to each eye 1
to 3x daily.
INJECTIBLE FORMS, e.g. for sub-cutaneoust intro-muscular or other
parenteral injection, including, in particular, intro-articular
injection, e.g. in the treatment of arthritic disease such as
rheumatoid arthritis, or for intro-lesional injection, e_,;~ in the
treatment of psoriasis.
Dispersions, suspensions, emulsions and like compositions suitable
for injection can be prepared in accordance with any of the
techniques known and generally employed in the art, for example by
dispersing, suspending or otherwise distributing Clclosporin in
fine particulate orthorhombic crystal form in an appropriate
liquid carrier medium with the addition of appropriate
emulsifiers, sufactants, stabilizing or flocculating agents etc...
Preferably the Ciclosporin component in such compositions will
have an average particle size of <100uM, more preferably <50uM,
most preferably <20uM. Suitably the average particle size will be
of the order of from 0.1 or 0.5 up to the indicated 20, 50 or
100NM maxima.
Where injectible forms are intended e.g. for sub-cutaneous or
intro-muscular injection, for example to achieve a depot affect,
use of larger particle size will be possible. Where such forms are
intended for intro-lesional application or, in particular,
intro-articular injection, use of finer particule preparations
will be indicated. For such uses a suitable average particle size
is e.g. of the order of from about 0.1 or about 0.5 to about 20uM,
e.g. ca. 5 to 15NM.
To achieve stability of particles in injectible forms, e.g. to
avoid increase in size of larger crystalline particles at the
expense of smaller particles, the particles will preferably be of
as uniform a size as possible. Suitably variation between maximum
and minimum particle size will not exceed about 50NM, more
preferably about 20uM.
.~ .... 1 3 41 3 9 6
- 23 -
Although the desired particle size may be achieved by use of
appropriate comminution procedures, e.g. micronisation, to
minimize particle size variation, to avoid the presence of
fragmented particles, to avoid modification of the crystal
surface, e.g. to retain wettability characteristics, and to more
easily meet required sterility requirements, it will generally be
preferred to employ non-comminuted microcrystals, i.e. crystals
grown to the desired size requirements as hereinbefore described.
Injectible compositions in accordance with the invention thus
appropriately comprise:
C3 a) Ciclosporin in fine particulate, orthorhombic, preferably
non-solvate orthorhombic, crystal form, e.g. CY-A/X-III, e.g.
as defined under any one of A to A', in particular A6 and A',
above, distributed or distributable within,
b) an injectible carrier medium.
Suitable carrier media include, in particular, pharmaceutical
grade water.
In addition compositions as defined under C3 will suitably~comprise
g) a sufactant as dispersant or emulsifying agent and/or a
flocculating agent, to assist or maintain distribution of the
particles throughout the carrier phase.
Suitable surfactant components (g) include polyoxyethylene-
-sorbitan fatty acid esters or polysorbates, e.g. as set forth
under (d3) above, e.g. Tween 80*or Polysorbate 80*(c.f. Fiedler,
2, p.p. 746). Suitable flocculating agents (g) include ethylene
diamine tetra-acetate and salts thereof, for example Calcium EDTA
and Sodium EDTA, as well as other equivalent chelating agents, and
peptisating agents such as succinic or citric acid.
In order to maintain particle size distribution, a stabilizing
agent is also suitably incorporated. Suitable stabilizing agents
*Trademark
~ 341396
- 24 -
include e.g. gelatins or modified gelatins such as the plasma
expander known and commercially available under the Trade Mark
Gelafundin and fiaemaccel or cold-water soluble gelatins of highly
purified collagen hydrolysates.
Compositions in accordance with C3 above may also include one or
more anti-microbial agents, e.g. as hereinbefore described, as
well as peptisating agents such as succinic or citric acid.
The ratio of components (a):(b) in compositions as defined under
C3 is suitably of the order of 1:10 to 1:1,000, preferably of the
order of from 1:50 to 1:200 e.g. about 1.100 p.p.w.
In order not to unduly disturb particle stability, surfactants
present as component (g) will preferably be present in only minor
concentrations, e.g. of the oder of from 0.1 to 2.0X, suitably ca.
1% by weight based on the weight of component (a). Where a
flocculating agent is present the ratio of flocculating agent to
component (a) is suitably of the order of from 1.3 to 1:8 p.p.w.
e.g. ca. 1:5 p.p.w. When a stabilizing agent is present, the ratio
of component (a):stabilizing agent is suitably of the order of
from 1:5 to 1:30, more preferably 1:10 to 1:30, e.g. ca. 1:20
p.p.w. When a peptisating agent is present, the ratio of peptising
agent to component (a) is suitably of the order of from 1:1 to
1:10, e.g. ca. 1:15 p.p.w.
The amount of Ciclosporin present in unit dosage forms for
injection in accordance with the invention will of course vary
depending on e.g. the condition to be treated, the site of
injection and the effect desired, e.g. whether depot effect or
relatively speedy release into the surrounding tissues is
contemplated. Suitably individual unit dosage forms will comprise
ca. 10 to 500 mg, especially from ca. 20 to 100mg
Ciclosporin/dosage.
M 1 341 39 6
- 25 -
As will be appreciated, unit dosage forms in accordance with the
invention also include more complex systems, e.g. double chambered
syringes or syringe barrels in which the particulate Ciclosporin
together with other appropriate components, e.g. stabilizer or
anti-microbial agent, is retained in a first chamber and the
carrier medium is retained in a second chamber, the components of
the two chambers being brought into admixture, e.g. following
actuation of the syringe plunger. Such double chamber syringe
systems are well known in the art and unit dosage foreis coatprising
them are to be understood as being within the purview of the
present invention.
ORAL DOSAGE FORMS e.g. for the treatment of auto-immune and other
diseases and conditions as hereinbefore set forth, e.g. for the
prevention of transplant refection.
Although, as previously indicated, oral dosage forms in accordance
with the present invention are generally of lesser interest and
hence less preferred, such forms are also within the purview of
the invention.
Oral dosage forms in accordance with the invention may be prepared
employing any appropriate carrier or carrier medium as known and
practiced in the art. Liquid or semi-solid oral dosage forms may
for example comprise Ciclosporin in orthorhombic crystal form,
e.g. as CY-A/X-III, together with pharmaceutically acceptable
diluents or carriers in which the crystal form is itself insoluble
or substantially insoluble. Such carriers and diluents include for
example trans-esterification products of natural vegetable oil
triglycerides and polyalkylene polyols as hereinbefore described
under (dl) for example Labrafils as well as reaction products of
natural or hydrogenated castor oil end ethylene oxide as
hereinbefore described under (d~), in particular liquid Cremophore
products, for example Cremophore EL.
1 341396
- 26 -
The Ciclosporin is preferably present in fine particulate form,
e.g. having an average particle size of from 0.5 to 200uH,
especially of the order of from e.g. 0.5. to 30uM, preferably less
than 20uM, e.g. from 0.5 to 10~M.
For convenience of administration, compositions as aforesaid will
suitably be filled into hard or soft-gelatin capsules.
Unit dosage forms in accordance with the invention for oral admi-
nistration suitably comprise from about 25 to about 75, or up to
200, e.g. ca. 50 or 100mg Ciclosporin/unit dosage. Such forms are
administered e.g. from 1 to 4x daily to achieve individual patient
serum ciclosporin concentrations (e.g. as determined by RIA) of
the same or equivalent order to those achieved employing
conventional Ciclosporin therapy, e.g. employing the available
Ciclosporin oral solution.
Ingredients referred to herein or defined as components of
pharmaceutical compositions in accordance with the invention are
to be understood as being suitable for pharmaceutical application
as required, e.g. in the case of ingredients of compositions for
oral administration, as being pharmaceutically acceptable or, in
the case of ingredients of compositions for topical
administration, as being topically, e.g. dermally or occulary,
applicable, acceptable or tolerable.
Further galenic forms for administration by equivalent or
alternative routes to those above described, will be apparent to
the skilled worker.
The following examples are illustrative of the present invention
in each of its various aspects.
1 34139fi
- 27 -
EgAtIPLE 1
PREPARATION OF Cy-A/X-III
a) 100 g of Ciclosporin in amorphous form are dissolved with stirring
in 400g polyethylene glycol 300 at 50°C. CY-A/X-III development
commences shortly after completion of dissolution. The solution is
allowed to stand for 24-48 hrs. at ca. 35-40 °C to complete
crystallisation and then diluted with 1000 ml water with vigorous
stirring. The obtained precipitate is separated by filtration,
washed with Hz0 to remove remaining polyethylene glycol and dried
to yield CY-A/X-III in substantially pure form: m.p. = i95 °C,
particle size = ca. 100-200 uM.
CY-A/X-III forms may be prepared analogously to the above
procedure but employing the following materials as solvent in
place of polyethylene glycol 300.
SOLVENT M.P. FOR OBTAINED CY-A/X-III (°C)
b) Polyethyleneglycol 300/
Cremophore RH 40
(1:1 by volume) ---
c) Cremophore EL 190
d) Solutol HS 15 188
e) Gelucire 44/14 T~ 180
f) Myrj 52 184
g) Tween 80 186
h) Cremophor RH40 190
In each case the obtained average particle size is of the order of
from about 50 to about 200 uM. Determined lattice structure is as
hereinbefore defined. X-ray diffraction data are as hereinafter
described with reference to Table III and Fig. IV.
S ,V
M.
1341396
- 28 -
EXAMPLE 2
PREPARATION OF CY-A/X-III
20g Ciclosporin as CY-AJX-I is dissolved in 60m1 ethanol and condensed
to 1/6th of the initial volume by distillation under reduced pressure
at >60°C. 160m1 polyethylene glycol (MW 300) + 20m1 H=0 are then added
slowly at SO-85°C. with stirring at 300 r.p.m.. An ultrasonic
vibrator/mixer is installed operating at 20,000 c.p.c. Nucleation is
initiated at 65-75°C. over 3 to 5 minutes. The obtained dispersion is
then cooled linearly over 10-20 minutes to 40°C. with continued
stirring at 700 r.p.m. and ultrasonic vibration. The obtained thick
pasty mixture is filtered, rinsed with polyethyleneglycol (MW 300)/Hz0
(3:1 p.p.vol), washed with warm water (30°C.) and dried at 50°C.
under
high vacuum, to yield CY-AJX-III in pure form : m.p. = 192°C. Particle
size = 3-lSUM. Determined lattice structure as hereinbefore defined.
X-ray diffraction data as described hereinafter with reference to
Table III and Fig. IV.
Repeat experiments indicate that crystal size is influence by
nucleation temperature and rate of crystal growth rather than duration
of processing. Thus by initiating nucleation at ca. 75°C. and
continuing crystallisation at the same temperature, an average crystal
particle size of ca. 50uM is obtained.
EXAMPLE 3
PREPARATION OF A GALENIC FORM FOR ORAL ADMINISTRATION
3.1 GELATIN ENCAPSULATED PREPARATIONS I
A suspension comprising 20 % by weight of Cy-A/X-IIi obtained in
accordance with the procedures of Example 1 in Gelucire 44/14 is
milled at elevated temperature employing a colloidal mill until the
average Cy-A/X-III particle size is of the order of from 0.5 to 10 uM.
1341396
- 29 -
250 mg portions of the obtained milled suspension are filled into
hard-gelatine capsules size 2, each comprising 50 mg Ciclosporin (as
Cy-A/X-III) as active ingredient.
3.2 GELATIN ENCAPSULATED PREPARATTON II
CY-A/X-III obtained in accordance with the procedures of Example 1 is
milled in a colloidal mill until the average GY-A/X-III particle size
is of the order of from 0.5 to 20uM. 50mg portions are then intimately
admixed with 2008 Cremophore EL and filled into hard gelatine capsules
size Z, each comprising 50mg Ciclosporin (as CY-A/X-III) as active
ingredient.
BBA~~PLE 4
PREPARATION OF GALENIC FORMS FOR TOPICAL APPLICATION
4.1 GEL PREPARATION I
Cy-A/X-III obtained in accordance with the procedures of Example 1 is
micronised employing an afr-Set mill. Micronised product having a
maximum particle size of 60-70uM and an average particle size of from
2-lOuM is combined and mixed in conventional manner with the
ingredients shown in the table below to provide an aqueous gel
comprising 10 % by weight Ciclosporin (as Gy-A/X-III) and suitable for
topical application, e.g. for the treatment of psoriasis.
INGREDIENT X BY WEIGHT
a) CICLOSPORIN (micronised Cy-A/X-III) 10.0
b) Polyacrylic acid (Carbopol 934P) 0.5
c) NaOH-solution (5%) 3.0
d) Methylparaben 0~07
e) Propylparaben 0.03
f) H20 (pharmaceutical grade) to 100.0 %
~ 34' 396
- 30 -
4.2 GEL PREPARATION II
INGREDIENT % BY WEIGHT
Composition A Composition B
a)CICLOSPORIN (micronised Cy-A/X-III) 10.0 1.0
b)Polyethylenglycol 300 30.0 30.0
c)Labrafil M 2130 CS 1.0 1.0
d)Carbopol 934 P 1.0 1.0
e)Sodium hydroxide 5% 6.0 6.0
f)Methylparaben 0.07 0.07
g)Propylparaben 0.03 0.03
h)Hz0 (p. g.) 51.9 60.9
100.0 % 100.0 %
a) is prepared as for 4.1 above. c), f) and g) are dissolved in b) with
warming and the obtained mix cooled to toom temperature. This solution is
then dispersed in water h). d) is added and the whole homogenized to effect
dispersion. a) is added and the mix again homogenized. e) is added to
neutralise, with stirring, and the resulting gel filled into collapsible
tubes for topical application, e.g. in the treatment of psoriasis.
4.3 OINTMENT PRAPARATION
INGREDIENT % BY WEIGHT
Composition A Composition B
a)CICLOSPORIN (micronised Cy-A/X-TII)1.0 14.0
b)Amerchol CAB 2.0 2.0
c)Liquid paraffin 42.0 37.5
d)White petrolatum 55.0 50.5
loo.o x loo.o x
~ 341 396
- 31 -
a) is prepared as for 4.1 above. b), c) and d) are melted together and
stirred and a) added at ca. 30°C, the particles being distributed
employing
an homogenizer. The obtained ointment is cooled and filled into collapsible
tubes for topical application, e.g. in the treatment of psoriasis.
4.4 CREAM PREPARATION
rn~r_urnr~nrr ~ av v~!TCUT
a)CICLOSPORIN (micronised Cy-A/X-IIT)1.0
b)Polysorbate 60 5.0
c)Cetylstearyl alcohol 10.0
d)Glycerol 85% 10.0
e)White petrolatum 24.0
f)Methylparaben 0.07
g)Propylparaben 0.03
h)Hz0 (pg)
49.0
100.0 %
a) is prepared as for 4.1 above, combined with components b) to h)
analogously to 4.3 above and filled into collapsible tubes for topical
application, e.g. in the treatment of psoriasis.
4.5 OINTMENT PREPARATION
A suspension comprising 20 % by weight of Cy-A/X-III (obtained in
accordance with the procedures of example 1) in polyethyleneglycol 300 is
wet-milled until the average Cy-A/X-III particle size is of the order of
from 2 to 10 uM. 10g of the obtained milled product is mixed in
conventional manner with 50g polyethyleneglycol 300 and 40g melted
polyethyleneglycol 1500 to yield a flowable paste comprising 10 % by weight
ciclosporin (as Cy-A/X-III) and suitable for topical application, e.g. for
the treatment of psoriasis.
1 341 39 6
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4.6 GEL PREPARATION
INGREDIENT x BY WEIGHT
a) CICLOSPORIN (micronised CY-A/X-III) 1.0
b) Labrafil M 1944 CS 1.0
c) Polyethylene glycol 300 30.0
d) Benzyl alcohol 1.0
e) Hydroxypropylmethylcellulose
(Methocel E4M)TM 2.5
f) HZO (p. g.) 64.5
100.0 %
a) is prepared as for 4.1 above. b), c) and d) are mixed, and f) added and
mixed. a) is then suspended in the resulting mixture by homogenisation. e)
is then added slowly with stirring until gel formation is complete. The gel
is then filled into collapsible tubes for topical application, e.g. in the
treatment of psoriasis.
4.7 GEL PREPARATION
INGREDIENT X BY WEIGHT
a)CICLOSPORIN (micronised CY-A/X-III) 5.0
b)Polyethyleneglycol 300 30.0
c)Glycerylmonostearate 5.0
d)Methyiparaben 0.07
e)Propylparaben 0.03
f)Carbopol 934 P 1.0
g)Sodium hydroxide 5% 6.0
h)HZO (p. g.) 52.9
100.0 %
B
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a) is prepared as for 4.1 above. c), d) and e) are dissolved in b) with
warming. The warm mixture is dispersed in h) by homogenisation and cooled
to room temperature. a) and f) are then added and the whole homogenised. g)
is then added and gel formation completed. The obtained gel is filled into
collapsible tubes for topical application, e.g. in the treatment of
psoriasis.
4.8 CREAM PREPARATION (WATER IN OIL)
INGREDIENT % BY WEIGHT
a) CICLOSPORIN (micronised CY-A/X-III) 2.0
b) Amerchol CAB 5.0
c) Miglyol 812 10.4
d) White petrolatum 53.0
e) H20 (p. g.) 30.0
100.0 %
a) is prepared as for 4.1 above and dispersed with b) in e) while warming
and homogenising. c) and d) are molten together, mixed and the mixture
added to the previously obtained dispersion with intensive homogenisation,
and the whole cooled to provide a fatty white cream. This is filled into
collabsible tubes for topical application, e.g. in the treatment of
psoriasis.
~.9 GEL/CREAM PREPARATIONS
Examples 4.1 to 4.8 above are repeated but employing the crystalline
product of example 2 directly as CICLOSPORIN component.
1 341 39 6
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EXAMPLE 5
5.1 INJECTIBLE FORM, E.G. FOR PARENTERAL APPLICATION
A Cy-A/X-III suspension is prepared under sterile conditions, by
conventional techniques and employing the following ingredients
INGREDIENT WT m
a) CICLOSPORIN (CY-A/X-III) 20.0
b) Polysorbate $0 4.0
c) Sodium carboxymethyl cellulose 5.0
d) NaCl 9.0
e) Benzyl alcohol 9.0
f) H20 (injection grade) to an end volume of 1.0 ml
The active ingredient release characteristics of the obtained formulation
are dependent upon the average particle sine of component a). Thus where
activity over longer periods of time is required, Cy-A/X-III product of
large average particle size (e.g. 100-200 uM as obtainable directly via the
procedures of example 1) will be chosen. Where activity over relatively
shorter periods of time is required, Cy-A/X-III product of relatively
smaller average particle size (e.g. obtained by micronisation or milling of
Example 1 product, e.g. to a particle sine of from 0.5-10 uM) will be
chosen. The obtained suspension is filled into injection ampoules for
parenteral administration or for intra-lesional injection in the treatment
of psoriasis, for example to provide a depot effect.
In an especially preferred embodiment the example is performed employing
CY-A/X-III prepared in accordance with example 2 above, having a particle
size of from 5-lSuM, and prepared under sterile conditions.
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5.2 INJECTIBLE FORMS, E.G. FOR INTRA-ARTICULAR TNJECTION
Injectible forms suitable for intra-articular administration are prepared
employing the following ingredients:
INGREDIENT WT
Composition A
a) CICLOSPORIN (CY-A/X-III) 10.0
b) Sodium carboxymethyl cellulose 10.0
c) Na EDTA 2.0
d) HZO (injection grade) to an end volume of 1.0 ml
Composition B
a) CICLOSPORIN (CY-A/X-III) 10.0
b) Polysorbate 80 0.1
c) Citric acid 2~0
d) H20 (injection grade) to an end volume of 1.0 ml
Formulation is carried out in accordance with standard techniques under
sterile conditions. Component (a) comprises non-comminuted micro-crystals,
produced under sterile conditions in accordance with the procedures of
example 2 and having a particle size of from 3-l5uM. The obtained
compositions are filled, again under sterile conditions, into ampoules for
injection, e.g. intra-articular injection for the treatment of rheumatoid
arthritis.
The advantageous properties of compositions in accordance with the
invention may be demonstrated in animal test models as well as in clinic.
Thus particular utility of compositions in accordance with the invention
intended for dermal application, e.g. in the treatment of psoriasis or
dermatosis, may be demonstrated in the following test model:
~ 341396
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ALLERGIC CONTACT DERMATITIS IN THE GUINEA PIG (DTH-TEST
Guinea pigs (Hartley, c~, 400-500 g) are sensitized by application of 50 ul
of 2 % dinitrofluorobenzene (DNFB) in acetone/olive oil (1:1) to the inner
side of the right ear. 7 days later, the ani:aals are re-treated with 20 ul,
0.5 % DNFB in acetone/olive oil (4:1) applied to marked areas on the
shaven, left and right flank. This second challenge exposure induces an
allergic inflammation, leading to reddening and cellular infiltration
(thickening) of the skin. Test composition in an amount of from 200-250 mg
is applied with a spatula to the DNFB treated area of the right flank. The
left flank is similarly treated with placebo as control. Application of
test composition/placebo is effected 5x at intervals of 20 mins, 8hrs., 24
hrs., 32 hrs., and 48 hrs., after the challenge. Skin thickness at the site
of application is determined before each application, and again 8 hrs.
after the last application, by raising the skin into a fold and measuring
the thickness of this. Degree of reddening or inflammation is also
estimated visually on a scale of from 0 to 4. Efficacy of test preparation
in preventing inflammatory response is determined by comparison with
results recorded for placebo treated flanks.
Results obtained in the above test model employing compositions in
accordance with examples 4.1 and 4.5 above but comprising 0.1 X by weight
Ciclosporin as CY-A/X-III, in comparison with placebo and with topically
applied 0.1 % Ciclosporin in convention oral solution form are shown in
Figs. I, II and III appendant to the present application. [A 0.1%
CY-A/X-III concentration is employed in this test model in view of its
extreme sensitivity. Test compositions are prepared entirely analogously to
examples 4.1 and 4.5 but by reduction of the Ciclosporin component to O.lg
and compensation by addition of 9.9g water in the case of example 4.1 and
polyethyleneglycol components in the case of example 4.5.)
In each figure, measured skin thickness is plotted against time. In Fig. I
results employing 0.1% (by weight) Giclosporin in conventional applied in
the form of the conventional, commercial oral, drink solution form are
compared with placebo. Slight but insignificant reduction in reaction as
compared with placebo is evidenced for the oral solution. In Figs. II and
1 341 396
_a,_
III, results employing 0.1% (by weight) Ciclosporin containing
compositions in accordance with examples 4.1 and 4.5 respectively are
compared with placebo. In both instances, substantial reduction in skin
thickening as compared with placebo are achieved following first
application and continuing through treatment as compared with placebo are
achieved following first application and continuing through treatment until
completion of the experiment.
Utility of compositions in accordance with the present invention may also
be demonstrated in clinical trials e.g. performed as follows:
CLINICAL TRIAL: TREATMENT OF PSORIASIS BX TOPICAL (DERMAL) APPLICATION
The trial is performed in randomized, placebo controlled, double-blind
design. Each patient receives active composition and placebo (vehicle
alone) on two symmetrical lesions simultaneously. Allocation of active and
placebo compositions is randomised to the left and right lesions.
Selected subjects are taken in groups of from 12 to 24 patients, aged 18-70
years including males and females who are postmenopausal, surgically
sterile or using oral contraceptives with a negative pregnancy test.
Inclusion criteria include: a) written informed consent and b) clinically
defined, stable, chronic plaque psoriasis. At least two moderate to severe
bilateral symmetrical lesions, each having an area of 4 to 25cm2 and a
severity score >5 on a scale ranging from 0 to 9. This score is a composite
of individual scores of 0 (least severe) to 3 (most severe) for each of
three parameters: erythema, scaling and skin thickness. Lesions on both
sides must be comparable within the same patient {i.e. not differing by
more than 20% in severity score or area).
The following exclusion criteria are applied:
a) Any medical condition which, in the opinion of the investigator, would
compromise the study.
. 1 341 39 fi
- 38 -
b) Systemic psoriasis therapy.
(PWA, methotrexate, steroids, retinoids, ciclosporin) within the last 2
months.
c) Specific topical treatment - except indifferent substances (e. g. white
vaselin) or formulations containing salicylic acid (not more than 10X) -
on lesions selected for study within the last 2 weeks before entering
the study. These emollients may however be used on lesions not selected
for the study.
d) Patients whose psoriasis appears to be spontaneously improving, flaring
without treatment, or rebounding from cessation of therapy.
e) Known hypersensitivity to any of the ingredients of the test medication.
f) Uncooperative patients who are unlikely to follow medical instructions
exactly, patients not willing or not able to attend regular visits.
g) Treatment with an investigational drug within one month before study
enrollment.
h) Severe concomitant disease, especially renal dysfunction with serum
creatinine above 1301~Mo1/1.
i) Hypertension (diastolic BP> 95mm Hg after 5 minutes in sitting
position). Patients with drug controlled hypertension are included into
the study.
j) Malignancy or history of malignancy, including skin cancer.
k) Acute bacterial, viral, or fungal associated skin lesions.
1) Pregnancy, lactation.
m) Patients with a history of alcoholism, drug abuse, psychosis, or
emotional or intellectual problems.
n) Patients who need continuous concomitant therapy with other
immunosuppressants, chemotherapeutic agents, compounds with a
nephrotoxic potential (e. g. aminoglycosides) and drugs which are known
to interact with the pharmacokinetics of Ciclosporin (ketoconazole,
erythromycin, phenytoin, phenobarbitone, rifampicin, INB, carbamazepine,
sodium valproate).
o) Patients with abnormal baseline laboratory values > 15X outside normal
limits for the testing laboratory.
p) Patients with clinically significant findings in physical examination.
"~.....
1 341 39 fi
- 39 -
Trial compositions employed are in accordance with any one of examples 4.1
to 4.9 hereinbefore described, e.g.: the 1X composition 8 of example 4.2 or
the lOX composition B of example 4.3.
Each patient receives a pair of 20g coded tubes bearing a two-panel label.
The label is blue for the substance which has to be applied on the right
side and red for the left side. Further, the side to be treated, left or
right, the study code, the investigator name, the patient number, and the
date of starting treatment, are written on each panel of the label, The
tear-off panel of the label is affixed in the case report form.
Test medication is applied 2x daily, morning and evening, the amount
applied depending on lesion size, to give a daily Ciclosporin dosaging of
from 0.2 to 2.0mg Ciclosporin/cm2, e,g. in one test series ca. 0.5mg
CiclosporinJcm2. Placebo is applied in equivalent amount. Neither trial
composition nor placebo is applied to a body surface >25cmz and no more
than 1g Ciclosporin is applied per week. At each clinic visit the
medication/placebo tubes are weighed and the topically applied daily dose
determined. Subfects may bathe shortly before application of medication.
Disposable gloves are put on and changed to apply each trial composition
and placebo to avoid cross contamination. At least three hours are allowed
to elapse between administration and washing of the treated areas. The
first application is supervised and a patient instruction sheet is
provided. Duration of treatment is 4 weeks. If complete clearance of
lesions occurs earlier, treatment is continued until week 4. Patients are
investigated before trial commencement (week -1), at the beginning of the
trial (week 0) and after 1, 2, 3 and 4 weeks,
Dermatological assessment (local psoriasis severity index or LPSI) is
assessed by the same investigator, not involved in medication, using the
following 0-3 numeric scales.
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Erythema Scaling Skin Thickness
0 = None 0 = none 0 = none
1 = Minimal: 1 = Minimal: 1 ~ Minimal:
faintly detec- poorly defined barely perceptible
table erythema; scales elevation
very light pink
2 = Moderate 2 = Moderate: 2 = Moderate:
dull red well-defined elevation clearly
distinguish- scales starting perceptible
able to pile up
3 = Strong: 3= Severe: 3 a Severe:
deep/bright well defined high elevation
red scales; piled
up to compact
The total LPSI range = value of erythema plus scaling plus thickness m 0 to
9 is evaluated. To enter inta the study, at least 2 signs must be moderate
to severe for both selected lesions (LPSI >5).
At entry and at each clinic visit, the area of the treated lesions is
evaluated (Entry size: G to 25cm= for each plaque). Area refers to presence
of one or more of the three criteria, erythema, scaling, thickening.
Pigmentation within the lesion area is not considered in area estimation
The plaques are registered by photographs before (week -1) and after the
treatment (week 4). Three pictures are taken: one showing both lesions
together and one for each lesion.
Pruritus is evaluated from patient interview using the following scale:
0 = none
1 = minimal: occasionally has urge to scratch
2 ~ moderate: has urge to scratch frequently
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3 -_- severe: has urge to scratch very frequently, may disturb sleep.
At end of the study, a global assessment of response to therapy is made in
accordance to the following criteria:
- cleared = 100% resolution
- marked improvement = at least 66% reduction of LPSI, and/or at least 66%
reduction of plaque area
- moderate improvement: between 65X and 33% reduction of LPSI, and/or
between 65% and 33% reduction of plaque area
- poor: less than 32% reduction of LPSI and/or less than 32X reduction of
plaque area
- worse: exacerbation of disease
Therapeutic success is defined as any lesion which has "cleared" or which
exhibits "marked improvement".
Physician and patient also indicate their preference for left or right side
medication in accordance with the following criteria:
. left side definitely better
. left side slightly better
. no difference detectable
. right side slightly better
. right side definitely better
To test efficacy at the end of treatment, subject LPSI scores and lesional
areas are compared by means of the WILCOXON-Sign Rank test for paired
treatments between Ciclosporin-treated sides and placebo treated sides with
respect to change in score from baseline to after 4 weeks of treatment.
All further comparisons (at other time points and for the remaining
parameters of efficacy), including the p-values attached to them, are taken
as descriptive of progress in trial.
In the above trial, subjects exhibit marked improvement in psoriatic
lesion, e.g. of LPSI scores for erythema, scaling and skin thickness, on
M. ~ 341396
" - 42 -
sides to which test composition comprising Ciclosporin is applied as
compared with side receiving placebo. Subjective improvement is also
recorded, together with improvement in recorded values for pruritis on
Ciclosporin treated as compared with placebo treated sides.
Positive results are also obtained in trials of parallel design for
subjects exhibiting contact dermatitis as diagnosed via 3 or more of the
following criteria:
- Pruritus
- Typical morphology and distribution:
Flexural lichenification or linearity in adults
Facial and extensor involvement in infants and children
-- Chronic or chronically-relapsing dermatitis
- Personal or family history of atopy (asthma, allergic rhinitis, atopic
dermatitis);
plus 3 or more minor features including:
Xerosis, ichthyosis/palmar hyperlinearity/keratosis pilaris, immediate
(type I) skin test reactivity, elevated serum IgE, early age of onset(< 5
years), tendency toward cutaneous infections (esp. Staph, aureus and Herpes
simplex)/ impaired cell-mediated immunity, tendency toward non-specific
hand or foot dermatitis, nipple eczema, cheilitis, recurrent
conjunctivitis, Dennie-Morgan infraorbital fold, keratoconus, anterior
subcapsular cataracts, orbital darkening, facial pallor/facial erythema,
pityriasis alba, anterior neck folds, itch when sweating, intolerance to
wool and lipid solvents, perifollicular accentuation, food intolerance,
course influenced by environmental/emotional factors and white
dermographism/delayed blanch. The trial period is three weeks, with
evaluation on a scale of 0 to 3 for the following parameters:
pruritis, erythema, exudation, excoriation and lichenification.
Topical formulations as hereinbefore described in examples 4.1 to 4.9,
containing 1-lOX Ciclosporin, for example the S.OX composition in
accordance with example 4.7 are also found to be effective in the treatment
~' _ 1 34139fi
- 43 -
of allergic contact dermatitis as well as of alopecia.
In accordance with the foregoing the present invention also provides:
D. A method of administering Ciclosporin to, or effecting Ciclosporin
therapy in, a subject receiving or requiring such therapy, which method
comprises administering to said subject an effective amount of
Ciclosporin in orthorhombic crystal form, in particular non-solvate
orthorhombic crystal form, e.g. as CY-A/X-III, e.g. as defined under
any one of A to A~ above.
In a more specific embodiment there is provided:
D1 A method of effecting immunosuppressive or anti-inflammatory therapy in
a subject in need thereof, which method comprises administering to said
subject an effective amount of Ciclosporin in orthorhombic crystal
form, in particular non-solvate orthorhombic crystal form, e.g. as
CY-A/X-III, e.g. as defined under any one of A to A~ above.
Purposes for which the above methods are applicable include treatment of
any disease or condition as hereinbefore described in relation to use of
Ciclosporin, e.g. prevention of transplant rejection, treatment of
auto-immune or rheumatic disease, treatment of any other disease or
condition involving undesirably high response of the immune system,
treatment of parasitic infection, treatment of chemotherapeutic resistance,
e.g. reversal of tumor resistance to anti-neoplastic chemotherapy, as well
as the treatment of alopecia. The above methods are in particular
applicable to the treatment of diseases or conditions of the skin or eye,
e.g. psoriasis, atopic or contact dermatitis, corneal transplant,
conjunctivitis and uveitis, by topical administration, as well as to the
treatment of rheumatoid arthritis or other disease or condition of the
joint, by intra-articular administration.
In the alternative the present invention provides:
E. Ciclosporin in non-solvate orthorhombic crystal form, e.g. as
' 341 39 6
- 44 -
CY-A/X-III, e.g. as defined under any one of A to A' above, for use as
a pharmaceutical, e.g. for use in the treatment of any disease or
condition as hereinabove set forth in relation to D or Dl above, for
example, for use as an immunosuppressive or anti-inflaamatory agent; as
well as
F. Ciclosporin in orthorhombic crystal form, e.g. as CY-A/X-III, e.g. as
defined under any one of A to A' above, for use in the preparation of a
pharmaceutical compostifon for use as defined under E above.
To assist identification of Cyclosporin in orthorhombic crystal form,
in particular CY-A/X-III, X-ray powder diffraction data for CY-A/X-III
and for the two forms CY-A/X-I and CY-A/X-II also referred to herein,
are provided in the following Tables I to III. Fig. IV attached
provides a representation of the X-ray powder diffraction diagram from
which the data in Tables I to III were obtained. The diffraction
diagram was obtained employing a Guinier-DeWolff II camera, using
CuKa-radiation, ~ = 1.542 A
1 341 X96
- 45 -
TABLE I
X-RAY DIAGRAM FOR GY-A/X-I
Line No. d (A) Intensity
1. 13.0 VS
2. 11.4 VS
3. 10.3 VS
4. 9.7 VS
5. 9.4 VS
6. 8.25 VS
7. 7.1 S
8. 6.1 M
9. 5.85 S
10. 5.6 S
11. 5.25 VS
12. 4.81 S
13. 4.58 S
14. 4.25 M
15. 4.0 M
16. 3.67 M
17. 3.45 M
.. 1341396
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TABLE II
X-RAY DIAGRAM FOR CY-A/X-II
Line No. d (fir) Intensity
1. 17.9 S
2. 11.4 VS
3. 11.0 S
4. 10.6 M
5. 10.2 S
6. 8.9 M
7. 8.7 M
8. 8.1 M
9. 7.2 VS
10. 6.3 M
ll. 5.95 M
12. 5.7 M
13. 5.35 M
14. 5.1 S
15. 4.85 S
16. 4.8 M
17. 4.55 M
18. 4.4 M
1 341 39 fi
_~7_
TABLE III
X-RAY DIAGRAM FOR CY-A/X-III
Line No. d (A) Intensity
1. 12.0 M
2. 10.4 VS
3. 9.6 S
4. 8.7 S
5. 7.9 M
6. 7,7 S
7. 6.7 M
8. 6.0 M
9. 5.83 S
10. 5.3 M
11. 5.2 M
12. 4.92 S
13. 4.88 S
14. 4.58 M
15. 4.48 M
16. 4.0 M
17. 3.59 M
18. 3.38 M
VS = very strong
S = strong
M = medium
