Testosterone 17β-N,N-dimethylglycinate hydrochloride: A prodrug with a potential for nasal delivery of testosterone

Article abstract of DOI:10.1002/jps.10083

The purpose of this study was to examine the potential of the nasal route for the systemic delivery of the poorly water-soluble drug testosterone (TS) using a water-soluble prodrug, TS 17β-N,N-dimethylglycinate hydrochloride. The physicochemical properties of the prodrug, in vitro hydrolysis in human liver homogenate, and in vivo nasal and intravenous experiments were performed in rats. The aqueous solubility of the prodrug was more than 100 mg/mL, compared with 0.01 mg/mL for TS, and its log partition coefficient between 0.05 M, phosphate buffer (pH 6) and octanol was 2.4. The prodrug was found to generate TS in 33% human liver homogenate and was absorbed from the nasal cavity rapidly and quantitatively. The bioavailabilities of both the prodrug and TS after nasal administration of the prodrug were similar to that after equivalent intravenous doses. These studies in rats suggest that this water-soluble prodrug of TS may have therapeutic utility for the management of TS deficiency.

Full text of DOI:10.1002/jps.10083

Testosterone 17b -N , N -D im ethylglycinate H ydrochloride: A
Prodrug w ith a Potential for N asal D elivery of Testosterone
3
4
5
AN W AR A.H U SSAIN , 1 AN SAM A.AL-BAYATTI, 2 AD N AN D AKKU RI, KAZU H IRO O KO CH I, M U N IR A.H U SSAIN
1College of Pharm acy, U niversity of Kentucky, Rose Street, Lexington, Kentucky 40536-0082
2Biochem istry D epartm ent, College of M edicine, U niversity of Baghdad, Baghdad, Iraq
3College of Pharm acy, Ferris State U niversity, Big Rapids, M ichigan 49307-2740
4Takeda Chem ical Industries, O saka, Japan
5Bristol-M yers Squibb Pharm a Com pany, D uPont Experim ental Station, W ilm ington, D elaw are 19880-0400
R eceived 20 M arch 2001; revised 4 O ctober 2001; accepted 24 O ctober 2001
Published online 22 February 2002
ABSTRACT: The purpose of this study w as to exam ine the potential of the nasal route
for the system ic delivery of the poorly w ater-soluble drug testosterone (TS) using a
w ater-soluble prodrug, TS 17b -N ,N -dim ethylglycinate hydrochloride. The physico-
chem ical properties of the prodrug, in vitro hydrolysis in hum an liver hom ogenate, and
in vivo nasal and intravenous experim ents w ere perform ed in rats. The aqueous
solubility of the prodrug w as m ore than 100 m g/m L, com pared w ith 0.01 m g/m L for TS,
and its log partition coef®cient betw een 0.05 M , phosphate buffer (pH 6) and octanol w as
2.4. The prodrug w as found to generate TS in 33% hum an liver hom ogenate and w as
absorbed from the nasal cavity rapidly and quantitatively. The bioavailabilities of both
the prodrug and TS after nasal adm inistration of the prodrug w ere sim ilar to that after
equivalent intravenous doses. These studies in rats suggest that this w ater-soluble
prodrug of TS m ay have therapeutic utility for the m anagem ent of TS de®ciency.
ß
2002
W iley-Liss, Inc. and the A m erican Pharm aceutical A ssociation J Pharm Sci 91:785±789, 2002;
D O I 10.1002/jps.10083
K eyw ords: testosterone; bioavailability; w ater-soluble prodrug; nasal absorption
IN TRO D U CTIO N
nate) given orally are sim ilarly m etabolized after
hydrolysis. C onsequently, TS and its esters are
generally adm inistered by intram uscular injec-
tion. In view of the ¯uctuations in serum TS
concentrations associated w ith TS esters, alter-
native m ethods for TS delivery such as buccal/
sublingual, nasal, and transderm al have been
investigated in search of a convenient and effec-
tive m odality of delivering the drug system ically.
R ecently, tw o transderm al TS patches have
becom e available com m ercially. A lthough this
route of adm inistration is effective, these patches
have several draw backs. The site of application in
one transderm al system , i.e., scrotum , and the
prerequisite special preparation of the site ren-
ders it undesirable to patients, resulting in a low
rate of com pliance w ith therapy regim ens. A lso,
Testosterone (TS) is considered to be the m ost
potent of the natural m ale sex horm ones. M en
w ith prim ary or secondary hypogonadism require
lifelong androgen replacem ent to prevent osteo-
porosis and m aintain norm al m uscle and bone
2
3
4
1
m ass, erythropoiesis, and sexual function. A l-
though TS adm inistered orally is readily absorbed,
it is ineffective because of extensive m eta-
bolism by the liver before reaching the system ic
circulation. E sters of TS (enanthate and cypio-
C orrespondence to: A . A . H ussain (Telephone: 859-257-
5939; Fax: 859-257-7585; E -m ail: aahuss1@ pop.uky.edu)
Journal of Pharm aceutical Sciences, V ol. 91, 785±789 (2002)
ß
2002 W iley-Liss, Inc. and the A m erican Pharm aceutical A ssociation
JO U RN AL O F PH ARM ACEU TICAL SCIEN CES, VO L. 91, N O . 3, M ARCH 2002
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786
H U SSA IN E T A L.
higher than norm al concentration of dihydro-
testosterone is reported due to the presence of
the enzym e 5a-reductase in high concentrations
4
in the scrotal skin. The other TS transderm al
system is intended for application to nonscrotal
5
skin. H ow ever, it is usually associated w ith skin
irritation and contact derm atitis at the applica-
tion site; som e patients discontinued using the
6
patches because of these rashes.
3
In a previous study by H ussain et al., TS w as
Figure 1. Structure of TSD G .
show n to be ef®ciently and com pletely absorbed in
rats intranasally w ith a half-life of elim ination
ꢀ40 m in. H ow ever, TS is poorly soluble in w ater
ꢀ0.01 m g/m L) and it is not feasible to prepare an
aqueous solution containing suf®cient drug
ꢀꢀ3 m g) for intranasal hum an use. Thus, if the
solubility of TS can be increased, it w ould
be an ideal candidate for nasal adm inistration.
O ne approach to achieve this objective is to use
solubilizers. B ut, this approach is not alw ays
successful because of the com plex interactions
betw een these agents and the nasal m ucosa that
m ay lead to tem porary or perm anent dam age to
the tissues. H ow ever, the use of a TS prodrug that
has an acceptable aqueous solubility is a m ore
practical approach for the form ulation of an
intranasal dosage form . In this report, it is show n
that a w ater-soluble ester of TS [TS 17b-N ,N -
dim ethylglycinate hydrochloride ꢀTSD G )] is
absorbed rapidly and com pletely w hen adm inis-
tered intranasally in doses that are unattainable
w ith TS itself. This prodrug w as selected over
other prodrugs carrying an am ine prom oiety, such
as dim ethylam inobutyrate, because it provided the
fastest hydrolysis rates in different tissues ꢀH us-
sain et al., unpublished data). A lthough conven-
tionally a com parison is m ade betw een a prodrug
adm inistered nasally and the parent drug intrave-
nously, it w as desired in these experim ents to
com pare the prodrug adm inistered nasally to the
prodrug adm inistered intravenously to better
assess its absorption and conversion potential.
R elatively high doses of the prodrug w ere used in
these studies in order to be able to m easure the
prodrug and TS by a high-perform ance liquid
chrom atography ꢀH PLC ) m ethod.
chloride w ere gently w arm ed at 408C until evolu-
tion of H C l gas ceased. N itrogen gas w as then
bubbled through the solution to rem ove unreacted
oxalyl chloride. The resulting acid chloride w as
dissolved in dim ethylform am ide and added drop-
w ise w ith stirring to a solution of TS in m ethylene
chloride. The reaction m ixture w as re¯uxed for
7
3 h. Sim ilar to w hat w as previously reported, the
ester w as isolated, converted to the H C l salt, and
characterized by elem ental analysis, nuclear m ag-
netic resonance ꢀN M R ), infrared, and H PLC .
E lem ental analysis ꢀC , H , N ) results w ere w ith-
1
in Æ 0.4% ofthecalculated values. H -N M R ꢀC D C l )
3
show ed the characteristic chem ical shifts of the
m ethylene group protons of the prom oiety, d 3.1
ꢀs, 2H ); and the N -dim ethyl protons, d 2.82 ꢀs, 6H ).
Stability of TS Prodrug 4TSD G )
in H um an Liver H om ogenate
A preparation of hum an liver hom ogenate ꢀ33% )
w as prepared by hom ogenizing slices of hum an
liver ꢀK eystone Skin B ank, E xton, PA ) in 0.05 M
phosphate buffer ꢀpH 6) at 08C . A TSD G solution
in the sam e buffer w as prepared at a 1 m g/m L
concentration and 0.1 m L of it w as added to 0.9 m L
of the liver hom ogenate and incubated at 378C . A t
different tim e points, 0.1 m L of the incubate w as
sam pled and added to 0.2 m L of acetonitrile. The
m ixture w as vortexed for 1 m in, centrifuged for
6 m in, and 20 mL of the supernatant injected onto
the H PLC colum n to analyze for TSD G and TS.
The experim ent w as run in triplicate. C ontrol
experim ents of only the prodrug in the buffer
show ed that the prodrug w as stable for the
duration of the experim ent.
M ATERIALS AN D M ETH O D S
Anim al Studies
M ale Sprague-D aw ley rats, each w eighing
approxim ately 300 g, w ere anesthetized w ith
pentobarbital sodium ꢀN em butal sodium solu-
tion; A bbott Laboratories, A bbott Park, IL)
ꢀ50 m g/m L using a 40 m g/kg dose intraperitone-
Synthesis of TSD G
1
The prodrug, TSD G , show n in Figure 1, w as
synthesized by a standard esteri®cation m ethod.
B rie¯y, dim ethylglycine hydrochloride and oxalyl
7
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A TE STO STE R O N E PR O D R U G FO R N A SA L D E LIV E R Y
787
ally). The anim als w ere prepared using a surgical
3
procedure allow ing intranasal adm inistration.
B rie¯y, an incision w as m ade in the neck, and the
trachea w as cannulated w ith a polyethylene tube.
A
closed-end tube w as inserted through the
esophagus to the posterior part of the nasal
cavity. The nasopalatine passage w as sealed w ith
an adhesive agent to prevent drainage of the drug
from the nasal cavity into the m outh. E ach rat
w as adm inistered 100 mL of a solution of the
prodrug in 0.05 M phosphate buffer ꢀpH 6) through
one nostril using a m icrosyringe. For intravenous
adm inistration, the sam e volum e w as delivered
into a cannulated jugular vein. Tw o dosing levels of
TSD G equivalent to 25 and 50 m g of TS/kg w ere
tested. E ach experim ent w as conducted in tripli-
cate. B lood sam ples ꢀ200 mL) w ere collected from a
cannula inserted in the fem oral artery at 0, 5, 10,
20,30,40,60,90,120,150,and 180m in.A fterim m e-
diate centrifugation, the plasm a w as separated and
analyzed by H PLC . A ll anim al procedures w ere
conducted according to the P rin ciples of L aboratory
A n im al C are ꢀN IH publication no. 85-23).
Figure 2. H ydrolysis of TSD G in 33% hum an liver
hom ogenate. D isappearance of the prodrug ꢀ.), and
appearance of TS ꢀ~). V alues represent average m olar
percentages.
genate at 378C . The apparent half-life of
hydrolysis of TSD G at this concentration of liver
hom ogenate w as estim ated to be 10 m in. The
quantitative liberation of TS from the prodrug
satis®es the basis prerequisite in prodrug design.
Analysis of TSD G and TS
A nalytical H PLC m easurem ents w ere conducted
using the follow ing: Spectro¯ow 400 solvent
delivery system ꢀK ratos, M anchester, U K ); Spec-
tro¯ow 77 absorbance detector ꢀK ratos), SP4270
integrator ꢀSpectra-Physics, M ountain V iew , C A );
R heodyne m odel 7125 injector, colum n: U ltra-
sphere octyl, 5 m, 250 Â 10 m m ꢀB eckm an, Full-
erton, C A ); guard colum n: ®lled w ith 25±37 mm
glass beads w ith chem ically bonded octadecyl
groups, 70 Â 2 m m ; injection volum e: 20 mL;
m obile phase: 0.1 M sodium acetate ꢀpH 4):
acetonitrile 50:50 v/v; ¯ow rate: 1 m L/m in; de-
tection w avelength 238 nm . R etention tim es for
TSD G and TS w ere 4.6 and 7.7 m in, respectively.
Solubility of TSD G and TS
D espite a relatively high partition coef®cient of
TSD G betw een octanol and 0.05 M phosphate
buffer ꢀpH 6), i.e., log P ˆ 2.4, the solubility of
TSD G in 0.05 M phosphate buffer ꢀpH 6) is m ore
than 100 m g/m L. The high solubility of this
prodrug is attributed to its surface activity, w ith
critical m icelle concentration of ꢀ3 m g/m L, sim i-
lar to that of a sim ilar prodrug, N ,N -dim ethyl-
9
am inobutyrate, previously reported. Log P of TS
is 3.410 w hereas its aqueous solubility is about
0.01 m g/m L. Such high solubility of the prodrug
m akes it feasible to form ulate aqueous solutions
containing a suf®cient am ount of TS for intra-
nasal adm inistration.
Solubility and Partition Coef®cient
The solubility of TSD G and TS in 0.05 M
phosphate buffer ꢀpH 6) w as determ ined. The
partition coef®cient of TSD G betw een 0.05 M
phosphate buffer ꢀpH 6) and octanol w as also
Pharm acokinetics: Intranasal and Intravenous
8
m easured follow ing standard procedures.
Figures 3 and 4 show the plasm a concentrations
of TSD G and TS after the intravenous adm inis-
tration of TSD G in doses equivalent to 25 and
50 m g of TS/kg, respectively. The apparent half-
RESU LTS AN D D ISCU SSIO N
Liver H om ogenate Study
life of TSD G disappearance ꢀ> 60% ), as deter-
Figure 2 show s the tim e course for the disap-
pearance of TSD G in 33% hum an liver hom o-
m ined from the initial slope, is 10 m in for the
low er dose and 20 m in for the higher dose,
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H U SSA IN E T A L.
Figure 3. Plasm a concentrations of TSD G ꢀ.) and
TS ꢀ~) after intravenous adm inistration of the prodrug
at a dose equivalent to 25 m g/kg of TS, m ean and SE
ꢀn ˆ 3).
Figure 5. Plasm a concentrations of TSD G ꢀ.) and
TS ꢀ~) after nasal adm inistration of the prodrug at a
dose equivalent to 25 m g/kg of TS, m ean and SE ꢀn ˆ 3).
w hereas the corresponding term inal elim ination
half-lives for TS are estim ated from Figures 3
and 4 be about 50 m in.
Figures 5 and 6 show the tim e dependence of
TSD G and TS concentrations after intranasal
adm inistration of TSD G in doses equivalent to 25
and 50 m g/kg of TS, respectively. It appears that
the prodrug is alm ost instantly absorbed and its
conversion into TS at the tw o dosing levels
com m ences im m ediately. The corresponding
term inal elim ination half-lives for TS are esti-
m ated from Figures 5 and 6 to be about 55 m in,
sim ilar to those observed after intravenous
adm inistration. H alf-lives of elim ination of TS
w hen adm inistered iv and nasally to rats w ere
3
also about 50 m in. The peak plasm a concentra-
tion of TS w as attained, both intranasally and
intravenously, w ithin 12 m in at the low er dose
and w ithin 20 m in at the higher dosing level.
In Table 1, the area under the curve ꢀA U C )
values for the prodrug and TS after intranasal
Figure 4. Plasm a concentrations of TSD G ꢀ.) and
TS ꢀ~) after intravenous adm inistration of the prodrug
at a dose equivalent to 50 m g/kg of TS, m ean and SE
ꢀn ˆ 3).
Figure 6. Plasm a concentrations of TSD G ꢀ.) and
TS ꢀ^) after nasal adm inistration of the prodrug at a
dose equivalent to 50 m g/kg of TS, m ean and SE ꢀn ˆ 3).
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Table 1. A U C V alues for TSD G and TS A fter Intravenous and N asal A dm inistration
of the Prodrug at D oses E quivalent to 25 and 50 m g/kg of TS
Intravenous
N asal
D ose, equivalent to 25 m g/kg of testosterone
A U C for TSD G ꢀmg Á h/m L)
1.65 Æ 0.33
1.77 Æ 0.12
1.63 Æ 0.41
1.76 Æ 0.06
A U C for TS ꢀmg Á h/m L)
D ose, equivalent to 50 m g/kg of testosterone
A U C for TSD G ꢀmg Á h/m L)
2.83 Æ 0.24
3.78 Æ 0.96
2.66 Æ 0.35
3.49 Æ 1.05
A U C for TS ꢀmg Á h/m L)
and intravenous adm inistration of TSD G at the
tw o dosing levels are show n. The sim ilarity in the
A U C values indicated that the prodrug w as
com pletely absorbed after intranasal adm inistra-
tion. A lso, the A U C for TS is alm ost doubled upon
doubling the dose of the prodrug intranasally or
intravenously. In addition to its potential for nasal
TS delivery, this prodrug m ay lend itself to
parenteral delivery replacing painful intram uscu-
lar injection of oily form ulations of TS derivatives.
episodic androgen release in hypogonadal m en. J
C lin E ndocrinol M etab 72:1054±1059.
3. H ussain A A , K im ura R , H uang C H . 1984. N asal
absorption of testosterone in rats. J Pharm Sci
73:1300±1301.
4. C unningham G R , C ordero E , Thornby JL. 1989.
Testosterone replacem ent w ith transderm al ther-
apeutic system s: Physiological serum testosterone
and elevated dihydrotestosterone levels. JA M A
261:2525±2530.
5. M eikle A W , M azer N A , M oellm er JF, Stringham
JD , Tolm an K G , Sanders SW , O dell W D . 1992.
E nhanced transderm al delivery of testosterone
across nonscrotal skin produces physiological con-
centrations of testosterone and its m etabolites in
hypogonadal m en. J C lin E ndocrinol 74:623±628.
6. A nonym ous. 1996. Testosterone patches for hypo-
gonadism . M ed Lett D rugs Ther 38:49±50.
7. M ilosovich SM . 1988. The utilization of prodrugs to
enhance transderm al penetration of testosterone,
deoxycorticosterone, and indom ethacin . PhD the-
sis, U niversity of K entucky, Lexington, K entucky.
8. E irkson C , H arras M C , O sborne C M , Sayre PG ,
Zeem an M . 1987. E nvironm ental assessm ent tech-
nical assistance handbook. Spring®eld, V A : R epro-
duced by the U .S. D epartm ent of C om m erce,
N ational Technical Inform ation Service.
CO N CLU SIO N S
The prodrug TSD G is w ater-soluble to an extent
that allow s the form ulation of drops, spray, or
som e other nasal dosage form , or even parenteral
dosage form s, in w hich the required dose for
hum ans is incorporated. Furtherm ore, it is hydro-
lyzed by hum an liver enzym es to TS rapidly and
quantitatively and thus it m ay have a potential
value in TS replacem ent therapy.
REFEREN CES
9. M ilosovich S, H ussain A , D ittert L, A ungst B ,
H ussain M . 1993. Testosteronyl-4-dim ethylam ino-
butyrate H C L: A prodrug w ith im proved skin
penetration rate. J Pharm Sci 82:227±228.
1. M atsum oto A M . 1994. H orm onal therapy of m ale
hypogonadism . E ndocrinol M etab C lin N orth A m
23:857±875.
2. Stuenkel C A , D udley R E , Y en SSC . 1991. Sub-
lingual adm inistration of testosterone-hydroxypro-
pyl-b-cyclodextrin inclusion com plex stim ulates
10. K im M I-K , Lee C -H , K im D -D . 2000. Skin perm ea-
tion of testosterone and its ester derivatives in rats.
J Pharm Pharm acol 52:369±375.
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