Note: Descriptions are shown in the official language in which they were submitted.
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Description
hPTH Fragment-(1-37), the Preparation Thereof, Medicaments Containing
Same and the Use Thereof.
The present invention relates to a hPTH fragment-(1-37), to fhe preparation
thereof,
to medicaments containing said fragment and to the use thereof.
Humane parathormone (hPTH), the hormone of the parathyroids, is a potentially
important therapeutic aid, e.g. for the treatment of osteoporosis and of
hypopara-
thyreoidism.
Osteoporosis (reduction in bone mass) (Riggs and Melto», N. E»gl. d. Med. 37
4, I 676-
1 G86, 198G) is a frequently occurring disease which mainly afflicts women in
the
menopause and elderly people. The persons affected suffer from, depending on
the
intensity, frequent fractures in the region of the backbone, the forearm or
thigh, pain
up to complete immobility, loss of fitness for work and of social contacts and
a
higher mortality risk. For the time being, healing is considered to be hardly
possible
(Co»se»sces Development Conference: Propl;ylaxis and Treatme»t of
Osteoporosis, 1987).
Experimental studies with animals (Selye, Endocrinology 7 G, 547-558, 1932;
Kalu et al.,
Lancet 1363-1366, 1970; Hefti et al., Cli». Science 62, 389-396, 1982; Tar» et
al.,
Endocrinology 110, 506-512, 1982; Podbesek et al., Endocrinolop~ 112, 17)00-
IOOG,
7983; Gunners-Hey and Hock, Metab. Bone Dis. & Rel. Rer. _5, 177-181, 1964) as
well
as more recent histological findings upon primary hyperparathyreoidism
(Delli»g et al.,
Kli». Woche»schr. 65, 643-G53, 1987) suggest that an increase in the bone mass
may
be accomplished by a treatment with PTH. Rccve et al. (Br. Med. d. 1340-1344,
1980) indeed achieved an improvement in the trabccular bone structure of
patients
suffering from osteoporosis by daily administration of small doses of PTH,
however
with a concomitant slight decrease in cortical bone mass. Recent findings
speak in
favour of that PTH, with a simultaneous administration of bone-active
substances such
as, e.g., 1,25-vitamin-Da (Slovik et al., d. Bone Ali»er. Rcs. 1, 377-381,
198G),
calcitonin (Hesch et al., Calcif. Tisme I»t. 44, 77G-180, 1989) or estrogen
(Reeve et
al., Proceedinb~s of the SIh International Co»bress on Bone Morphometry,
Niib>ala, duly 24-
29, 1988) increases the bone mass of the Spongiosa without Corticalis loss.
Hypoparathyreoidism (PTH deficiency) (Kruse, Monatsschr. Ki»derheilku»de 13G,
G52-
GGG, 1988) will occur either congenitally or as a consequence of surgery or of
radiation treatment in the cervical region and results in a decreased calcium
con-
centration in blood. The patients fend to have conwlsive COs. if the PTH
dericicncy
does already exist during infancy or childhood, a reduced mental development
and a
defect tooth and bone development are imminent over a long term. While a
therapy
with calcium and/or vitamin D preparations will in most patients normalizd the
Translation from PCT/EP90/01807
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calcium concentration in scrum, il goes along witla an increased risk of
kidney
damage. This risk of a medicamentous treatment can be avoided by a hormone
substi-
tution therapy with PTH.
Eventually, it has most recently been shown that the parathyreoid hormone
exhibits
an antihyyperlensive activity (Nickols, Blood Vessels> 24, 720-124, 1987).
In the treatment of osteoporosis and hypertonia as well as in the hormone sub-
stitution in the case of hypoparathyreoidism, the PTH must be regularly
administered
over an extended period of time, if necessary lifelong. Therefore, the PTH
administer-
ed must be free from impurities and must not induce the formation of
antibodies.
This requirement can be most efficiently met by peptides with the amino acid
sequence of human PTH which have been synthesized via the chemical rouse or by
genetic engineering. The secreted PTH molecule consists of 84 amino acids
[hPTH-(1-
84)~. However, peptides of this order of magnitude are difficult to chemically
synthe-
size and may be more easily prepared by genetic engineering.
Hitherto, two different peptides having the amino-terminal partial sequences
of the
human PTH - the hPTH-(1-34) and the hPTH-(1-38) - have been synthesized. In
the clinical tests under the regimen of one single injection for a diagnostic
applica-
tion, the short-term effects expected from previous experience with extracted
bovine
PTH (bPTH) were observed with hPTH-(1-34) (Mallette et al., J. Cli». E»docri».
Metab. G7, 964-972, 1988) as well as with hPTH-(1-38) (Kr»se and Kracht, Eur.
J.
Pediatr. 146, 373-377, 1987), i.e. the temporary stimulation of the excretion
of
phosphate and cyclic adenosine monophosphate (CAMP) in the urine and a
temporary
increase of the CAMP concentration in the plasma.
In the therapeutical test of said peptides with a small number of osteoporosis
patients, with hPTH-(1-34) (Reeve ct al., Pracecdi»~s of the Srh I»tcr»atio»a1
Congress v»
13o»e Moryhometry, Niigata, July 24-29, 1988) as well as ~~ith hPTI-I-(1-38)
(Hesch ct
al., CaJcif. Tissue I»t. 44, 17G-18(l, l X89) some succtas could he achieved.
The disadvantage of the PTI-I fragments so far available, however, is that in
sornc
patients they provoke the formation of antibodies which may reverse the effect
provided by the exogcneously supplied fragments of even of the endogenous P'TI-
I
[cf., for example, for hPTH-(1-34) A»dra» et al., J. Cliu. E»docri». Metab.
G4, 937-
943, 1987, and for hPTH-(1-3$) St~iyrnan» et al., A~onatssc.Hr. Ki»derheilk.
736= 107,
1985.
About twenty years ago, Berson and Yalow (J. Cli». E»docri»ol. Merab. 28, 7037-
1047, 1968) furnished evidence of that various PTH fragments occur in human
plasma which could have been formed either by a fast peripheral degradation of
the
total molecule or by secretion of P-fI-I fragments. It was shown that the
products, the
amounts of which prevailed in the circulation, of the peripheral pTH
metabolism are
large carboxy-terminal fragments having no biological activity which are
formed in the
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liver (cf., for example, .(~'Amoztr a»d Huef, Am. J. Physiol. 246, E249-255,
7984). I~ue
to the experimental results available it was assumed that upon an intact renal
function
the hP'I'H-(1-84) is the dominating biologically active P'1'1-I form. In
cotttrast, at a
restricted renal function, there appeared a clearly visible peak in the
elution position
of the hPTH-(1-34) in fractionated plasma of the respective patients (cf.
Gru»baum et
al., Ant. J. P)zysiol. 247, E442-448, 1984).
The existence of a biologically active FTH fragment circulating in human
plasma of healthy patients could so far not be detected.
Thus, various attempts have been made to simulate the endogenous decomposition
of
the PTH by in vitro investigations, thereby to draw conclusions with respect
to primary
cleavage positions in the total molecule. In this context, nearly each of the
positions
between the amino acids No. 5 and No. 43 of the amino-terminal end of the
chain
has been contemplated (cf., for example, Barli»g et al., I»t. J. BiocJte»t.
16, 815-821,
1984). Ztzll and Chua»g (J. Biol. Che»t. 260, 1608-1 G13, 1985), among others,
carried
out investigations with bPTH and cathepsin D. They were able to prove that
upon the
enzymatic decomposition of bovine PTI-I (bPTH) with the enzyme derived from
bovine
spleen the C-terminal fragments (35-84) and (38-84) and the complemental N-
terminal fragments (1-34) and (1-37) are formed. The bPTH described in that
paper
is distinguished from the fragment hPTH-(1-37) found by us in the positions 1
(Ser
instead of Ala), 7 (Leu instead of Phe), and 16 (Asn instead of Ser). The last-
mentioned bovine fragments proved to be biologically active in in vitro
investigations
with kidney membranes of rat and bovine origin, while, however, the fragment-
(1-37)
was detectable only in very low amounts and was fast hydrolyzed to form the
fragment-(1-34). The authors concluded therefrom that the final product of the
enzymatic decomposition with cathepsin D of PTH in the bovine animal is the
fragment-(1-34). In contrast thereto, other authors doubted that in vivo any N-
terminal fragments would occur at all in the plasma (cf., for example,
Golt~man» et
al., J. C1i». I»vest. 65, 1309-1317, I980). Thus, no biologically active N-
terminal PTI-I
fragments formed by a peripheral degradation could be identified in normal
rats
(Bri»gluusl et aL, Ant. J. Ph~~siol. 255, E886-893, 1982). MacGreb~or et al.,
(J. Biol.
Che»t. 261, 1929-1934, 1981) found that bovine parathyroids in culture do not
secrete any biological active N-terminal PTH fragments. In summary, these
findings at lhc date of lhc present invention suggest that no biologically
active N-terminal PTI-I fragments circulate in human or animal blood
plasma.
It is the object of lhc present invention to provide a new hPTH fragment-(1-
37)
which is a well accessible medicament having the biological and therapeutic
activity of
natural parathormone (PTH), which at least has the efficiency of the knov~m
hPTH
fragments (1-34) and (1-38) and avoids the drawbacks inherent thereto - and
especially the induction of the antibody formation.
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It is a further object of the present invention to provide a preparation
method for
said hPTH fragment-(1-37) and the use thereof as medicament for various
therapeutic and diagnostic indications.
Said objects are attained by a hPTH fragment comprising a novel amino acid
sequence.
Thus, the present invention relates to
a hPTH fragment-(1-37) having the amino acid sequence
Ser - Val - Ser - Glu - Ile - Gln - Leu - Met - His - Asn - Leu - Gly - Lys
- His - Leu - As» - Ser - Met - Glu - Arg - Val - Glu - Trp - Leu - Arg -
Lys - Lys - Leu - GI» - Asp - Val - His - As» - Phe - Val - Ala - Leu
and its natural and pharmacologically compatible derivatives, especially
amidated,
acetylated, phosphorylated and glycosylated hPTH-(1-37) derivatives.
The present invention further relates to a method for preparing said hPTH
fragment-
(1-37) or the derivatives thereof, said method being characterized in that
said
fragment is prepared via a prokaryotic or an eukaryotic expression and is
purified by
means of chromatography, and to a further method for preparing the hPTH
fragment-
(1-37) or the derivatives thereof by isolating said fragment from human blood
by
chromatographic procedures in a per se known manner, and eventually to a
method
for preparing the hPTH fragment-(1-37) or the derivatives thereof by preparing
said
hPTH fragment by the conventional methods of solid phase and liquid phase
synthesis
from the protected amino acids contained in the sequence as set forth,
deblocking it
and purifying it by means of the established chromatographic procedures.
According to the invention it has surprisingly been found that the shortest
hPTH
fragment exhibiting full biological activity, which is formed by the primary
cleavage of
the hPTH-(1-S4) in the human body, is the hP1'1-I-(I-37) (cf. Example 1). It
has
also surprisingly been found that the spatial structure of hI'Tl-I-(1-37) is
clearly
distinguished from those of the fragments hitherto known (cf. Example j),
which fact
points to its specific structure-effect relations and antigen properties.
The hPTH fragment-(1-37) has been chemically synthesized (cf. Example 2) and
has
been formulated as a medicament. Also the preparation by genetic engineering
using
conventional vectors has been elaborated: Via the genetic engineering route
the hPTH-
(1-37) peptide is prepared (1) in prokaryotic organisms as well as (2) in
eukaryotic
organisms. Por the prokaryotic expression, we prefer to utilize Escherichia
coli.
Available for this purpose are, among others, expression vectors for the
secretory
expression (r.g. pSPti, pRit-derivatives, Pharmacia), for the direct
cytoplasmic
expression (e.g. pKK-derivatives, Pharmacia) or expression as fusion protein
(pMC1871, Pharmacia) (literature see in Marsto» et al., Biochem. I. 240, 1-12,
198G).
For the eukaryotic expression we are able to make use of various organisms and
Translation from PCT/EP90/()18U7
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vectors, e.g. insect cells (Summers and S»ritlr, Tes. Abric. Ez7~. Strr.
(brrll) .1555, 1987),
yeasts (Hitaema»» et ai., Nature 293, 717-722, 7981), filamentous fungi
(Melton et al.,
Proc. Natl. Acad. Sci. USA, 81, 1470-1474, 1984) and mammal cells (Zettlmeissl
et
al., Biotecla»ology _5, 720-725, 1987), among wich we prefer to use the insect
cells.
The expressed peptide is purified by methods of chromatography, preferably as
set
forth in Example 1.
The medicament formulation contains hPTH-(1-37) or a physiologically
compatible
salt of hPTH-(1-37). The form and composition of the medicament containing the
hPTH-(1-37) depends on the kind of administration. Human hPTH-(1-37) may be
administered parenterally, intranasally, orally and by vvay of an inhalation.
It is
preferred to formulate hPTH-(1-37) into a preparation for injection, either as
a
solution or as a lyophilizate to be dissolved immediately before use. The
medicament
formulation may further contain auxiliary materials which are desired or
necessary to
meet the requirements of dispensing, to contribute to the solubility,
stability or sterility
of the medicament or to increase the efficiency of the resorption into the
body. The
daily dose to be administered depends on the indication. In the therapy of
osteo-
porosis by i.v./i.m, injection, the daily dose is within the range of from 100
to 1,200
units (ft.g)/day, while for a daily subcutaneous injection it is preferably
30U to 2,A00
units (li.g)/day. The determination of the biological activity is based on
measurements
against International reference preparations and reference preparations by our
labor-
atory for human PTH fragments in a common biological assay for hPTH fragments.
The fragment according to the invention hPTH-(1-37) is particularly suitable
as a
long-term therapeutic for hypoparathyreoidism and osteoporosis, because it
exhibits an
excellent biological activity and, on the other hand, does not induce any
immuno-
reaction even in the case of a life-long treatment.
The preparation according to the invention is further suitable as a blood
pressure-
slabilizing agent for a long-term or permanent treatment of essential
hypcrtonia.
The preparation according to the invention is further to he applied as an
agent for
the therapy of renal diseases, in the intensive care, and for the therapy of
lung
diseases (cf. Example G).
The preparation according to the invention is further to be employed as an
agent for
the therapy of male impotence (cf. Example G).
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The invention is further illustrated by means of Examples and of the
Figures to which reference is made in the Examples.
Figure 1: Sephadex G 25 - Preparative large-scale gel chromatography of the
alginic acid eluates for a rough separation by molecular weights and for
de-salting the crude peptide extract. The hPTH fragments determined by
RIA are found in the hatched area.
Column: Pharmacia K 100/100; TD 10 cm x 80 cm
Material: Sephadex G 25 medium
Eluant: 1 M Acetic acid
Flow Rate: 5 ml/min
Absorption: 280 nm
Sephadex is a Trademark.
Figure 2: Preparative HPLC cation exchanger chromatography for the further
separation of the peptide material from Figure 1. The fractions No. 2-5
of 16 ml each contain more than 640 pmoles each per fraction of hPTH-
(44-68)-IR material (hatched).
Column: HPLC steel column 2 cm x 10 cm
Material: Parcosil Pepkat
Eluant: A: 5 mM K.,HP04 pH 3.0; B: like A in 1 M HCl
Flow Rate: 8 ml/min
Absorption: 280 nm
Gradient: 0-60 % B in 60 min
Parcosil Pepkat is a Trade~nark_
Figure 3: Semi-preparative RP-chromatography for separating the fractions No.
2-5
of Figure 2. In the fractions No. 17 and No. 20 of 3 ml each there are
present more than 180 pmoles of hPTH fragments detectable by RIA for
hPTH-(44-68).
Column: HPC steel column 1 cm x 10 cm
Material: Orpegen RP HD-gel 7/300
Eluant: A: 0.01 M HCI; B: like A in 80% acetonitrile
Flow Rate: 3 ml/min
Absorption: 280 nm
Gradient: 0-60% B in 60 min
Temperature: 45 ° C
Orpegen is a Trade-mark.
Figure 4: Work-up of , fraction 20 from Figure 3 by means of semi-preparative
canon exchanger chromatography. Fractions .32 and 33 of 10 ml each
contain 7~0 pmoles and 600 pmoles, resoectively, of a hPTH fragment
which is detectable by the RIA for hPTH-(44-68).
Column: HPLC steel column 1 cm x 5 cm
Material: Parcosil Pepkat
CA 02071538 1999-11-02
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Eluant: A: SmM K2HP04 pH 3.0; B: like A in 1 M NaCI
Flow Rate: 3 ml/min
Absorption: 230 nm.
Gradient: 0-50% B in 50 min
Figure 5: Intermediate Step 1: Analytical RP chromatography of the freactions
32
and 33 of Figure 4. Two molecule forms of hPTH-(44-68)-IR are detect-
able, namely in fraction 12 of 3 ml an amount in excess of 150 pmoles
and in the fractions 15 and 16 of 3 ml amounts in excess of 300
pmoles in each.
Column: HPC steel column 1 cm x 10 cm
Material: Orpegen RP HD-gel 7/300
Eluant: A: 0.1 % TFA; B: like A in 80% acetonitrile
Flow Rate: 3 ml/min
Absorption: 280 nm
Gradient: 0-40% B in 60 min
Temperature: 4~ ° C
Figure 6: Intermediate step 2: Analytical cation exchanger chromatography of
the
fractions 15 and 16 of Figure S. The hPTH-(44-68)-IR substance is
particularly concentrated in fraction 9 of 2 ml (in excess of 200 pmoles).
Column: HPLC steel column 0.5 cm x S cm
Material: Parcosil Pepkat
Eluant: A: SmM K2HP04 pH 3.0; B: like A in 1 M NaCI
Flow Rate: 0.7 ml/min
Absorption: 230 nm
Gradient: 0-50% B in 50 min
Figure 7: Hydrophobic interaction chromatography of the fractions 8 and 9 of
Figure 6. The fractions Nos. 5-10 of 0.87 ml each contain the hPTH-(44-
68)-IR material in concentrations of more than 170 pmoles/fraction.
Column: HPLC steel column O.Scm x 5 cm
Material: Parcosil Pro HIC
Eluant: A: 100 mM Na2HP04 pH 6.5;
B: like A in 3M (NH4)~S04
Flow Rate: 0.7 ml/min
Absorption: 230 nm
Gradient: 100-0% B in 45 min
Paroosil Pro is a Trademark.
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Figure 8: Analytical RP chromatography of the fractions 5 to 10 of Figure 7.
The main peak is observed with the hPTH-(44-68)-IR.
Column: HPLC steel column 0.5 cm x 5 cm
Material: Orpegen RP HD-gel 7/300
Eluant: A: 0.1 % TFA; B: like A in 80% acetonitrile
Flow Rate: 0.7 ml/min
Absorption: 230 nm
Gradient: 0-40% B in 60 min
Temperature: 45 ° C
Figure 9: Analytical RP chromatography for the final purification of the hPTH
frag-
ment. The material of fraction 25 from Figure 8 was re- chromatograph-
ed, and in the main peak there results a highly pure peptide, the
sequence of which was determined to be hPTH-(38-84).
Column: HPLC steel column 0.5 cm x S cm
Material: Orpegen RP HD-gel 7/300
Eluant: A: 0.1 % TFA; B: like A in 80% acetonitrile
Flow Rate: 0.7 ml/min
Absorption: 230 nm
Gradient: 0-40% B in 60 min
Temperature: 4~ ° C
Figure 10: Reversed-phase HPLC chromatography of the synthetic hPTH fragments
from the N-terminal range, namely hPTH-(1-33), hPTH-(1-34), hPTH-(1-
37), hPTH-(1-38). If these fragments are used as reference for natural
fragments of the N-terminus of hPTH circulating in blood, then it can be
shown that the hPTH-(I-37) is the correct molecule form in the human
blood.
Column: Parcosil ProRP 300 - 7, C4, 125 x 4 mm
Temperature: 55 ° C
Eluant: A: 0.1% trifluoroacetic acid
B: like A + 80% acetontrile
Gradient: Start: - 25% B
5~ min - 50% B
60 min - 100% B
Absorption: 230 nm
Flow Rate: 0.7 ml/min
Parcosil ProRP is a Trade~aark.
Figure 11: Time dependence of the intracellular cAMP concentration of PC-12
cells
after the stimulation with hPTH fragments. 1.5 x 105 cells were incubated
with 10-7M hPTH-(1-33), hPTH-(1-37), and hPTH-(1-38) in the
presence of 10-4M IBM X for 0, 2.5, 10 and 20 minutes. For control,
the cells were incubated with only IBMX for 20 minutes. The values are
average results of triple experiments ~ S.D.
~'~1~.'~
1U
Figure 12: Dosis-effect relation between the concentrations of hPTH-(1-33),
hPTI-I-
(1-37), and hPTH.-(1-38) and the intracellular cAMP level in PC-12 cells
after S minutes of stimulation. 2.2 x 105 PC-12 cells were incubated each
with 10-11 (double run), 10-1~, 10"9, 10-$ and 10-~M of each of hPTH-
(1-33), hPTH-(1-37), and hPTH-(1-38) in triple experiments in the
presence of 10-4M IBMX for 5 minutes, and the intracellular eAMP con-
centration was measured. The IBMX control values without PTH
fragments were 18.0, 26.1, 20.9. The values depicted are average values
~ S.D.
Figure 13: UV circular dichroism spectra of the PTH fragments hPTH-(1-33),
hPTH-(1-37), and hPTH-(1-38). In comparison to other hPTH frag-
ments, the hPTH-(1-37) has a particularly prominent structure.
Figure 14: Purification of the chemically s)mthesized hPTH-(1-37) in the first
step.
Here, a canon exchanger chromatography was carried out for the crude
product. According to the reference, the synthetic hPTH-(1-37) appears
in the higher peak at a retention time of 29 min (arrow).
Column: Parcosil PepKat 3UU - 7, 125 x 4 mm
Temperature: 25 ° C
Eluant: A: 5 mM NaH2P04
B: 5 mM NaH2P04, 1M NaCI, pH = 3.U
Gradient: Start: - S% B
57 min - 100% B
Absorption: 230 nm
Ftow Rate: 1 ml/min
Figure 15: Reversed-phase HPLC chromatography of the pre-purified material of
Figure 14 on a semi-preparative scale. A main peak eluting from
22.SU min is obtained which corresponds to the reference of hPThl-(1-
37).
Column: Parcosil ProRP 30U - 7, C4, l0U x 2U mm
Temperature: 25 ° C
Absorption: 230 nm
Eluant: A: U.1% trifluoroacctic acid
B: like A + 80°~o acctontrilc
Gradient: U-100°!o B in 60 min
Flow Rate: 7 ml/min
Figure 16: Reversed-phase HPLC chromatography of the material of Figure 15
with
the representation of the final purification. The peak corresponds to the
reference of chemically synthesized hPTH-(1-37).
Column: Parcosil ProRP 300 - 18, C4, 125 x 4 mm
Temperature: 25 ° C
Absorption: 230 nm
Translation from PCT/EP90/01807
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Eluanl: A: 0.1% trifluoroacetic acid
B: like A + 80% acetontrile
Gradient: 0-lU0% in 60 min
Flow Rate: 0.7 ml/min
Figure I7: Plasma desorption mass spectrum of the synthetic hPTH-(1-37) from
the
preparation of Figure 16. The molecular weight found of MW 4398.1 is
identical with the theoretically calculated mass of MW 4401.0 of the
molecule within an error range of <0.1%. Side sequences are not visible.
The peak at MW 2201.5 corresponds to the double-ionized form, also
within the limits of error of the measurement. This mass determination
was confirmed by counter-sequencing.
EXAMPLE 2
Indirect Determination of the Sequence of a
Circulating, Biologically Active PTH Fragment
The starting material employed was a hemof~ltrate which is obtained in large
amount
upon the treatment of patients suffering from renal insufficiency and contains
all
plasma constituents up to a molecular size of about 20,000 Dalton.
I. Recovery of the crude peptide material
The hemofihrate was recovered by means of a hemofiltration apparatus from the
company Sartorius using cellulose triacetate fillers having an exclusion size
of
20,000 Dalton (Type SM 40042, Sartorius, Gottingen, Germany). The filtrate was
derived from renal insufficiency patients who were in a stable metabolism
condition
due long-term hemofiltration. 1000 liters of hemoflltratc were recovered and
immediately after the recovery protected against proteolytic decomposition by
flow-
heating, acidifying and addition of enzyme inhibitors. Then a crude peptide
fraction
(about 100 g) was isolated by extraction with alginic acid according to the
method of
Forssmann disclosed in the DE 36 33 797 Al.
II. Isolation of a fraction (Code 51.5) exhibiting immunorcactivity in mcdium-
regional- and C-terminal-specific radioimmunoassays for parathormonc
(PTFI)
1. De-salting the crude peptide material through Scphadcx G-25
Tlxe crude peptide fraction obtained from the alginic acid extraction was
subjected to
a l;el-chromatic separation at 8 °C on a column filled with Sephadex G-
25
(Pharmacia, Uppsala, Sweden) equilibrated with 1M acetic acid (Fig. 1).
Translation from PCT/EP90/01807
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T'he immunorcactivity in the Pools I through IV was measured in a
radioimmunoassay
(RIA) for human P'I'I-I with medium-regional specificity [hP'I H-(44-68)-RIA,
company Immundiagnostik, Darmsladt, Germany]. According to the data provided
by
the producer, the lower limit of detection is 6 fmoles/test [six femto-moles
per test]
of the standard employed [hPTH-(44-68)]. Intro-Assay and Inter-Assay variation
coefficients are from 10 to 13% and from 12 to 21%, respectively. The
synthetic
hPTH peptides hPTH-(i-34), hPTH-(28-48) and hPTH-(64-84) do not show any
cross-reaction, while the peptides hPTH-(53-84) and hPTH-(1-84) produce 100%
cross-reaction in the hPTH-(44-68)-RIA.
Pool I contained the total immunoreactivity measured in the hPTH-(44-68)-RIA,
whereas no immunoreactivity was detectable in the other fractions.
Pool 1 also contained the highest C-terminal immunoreactivity, measured in the
hPTH-(53-84)-RIA (company Immundiagnostik, Darmstadt, Germany). The hPTI-i-(53-
84)-RIA, according to the data provided by the producer, has a lower limit of
detect-
ion of 4 fmoles/tesl of the standard employed [hPTH-(53-84)]. The Intro-Assay
and
Inter-Assay variation coefficients are from 8.3 to 9.8% and from 11 to 14%,
respectively. The synthetic hPTH peptides hPTI-I-(1-34), hPTH-(28-48) and hPTH-
(44-68) do not show any cross-reaction, while the peptides hPTH-(64-84) and
hPTH-
(1-84) produce 100% cross-reaction in the hPTH-(53-84)-RIA.
Pool I (hatched) was employed for the further isolation, because here, due to
measurements in two PTH-RIA, the highest concentration of C-terminal PTH
fragments occurred.
2. Preparative cation exchanger chromatography of Pool I
A further preparative separation was effected at room temperature on a canon
exchanger column (Fig. 2). A total amount of about 6 g of the material of Pool
I
was separated in 12 identical chromatographic runs, and the immunoreactivity
of was
monitored in the hPTH-(44-68)-RIA (Fig. 2). The fractions Nos. 2-5 (hatched)
not
shown in the Table contained the highest irnmunorcactivity in the hPTI-I-(44-
68)-RIA
and were pooled for further separation. They also contained the highest
immunoreactivity in the hPTI-1-(53-84)-RIA.
3. Semi-preparative reversed-phase chromatography of the immuno-
rcactivc Pool from the preparative canon-exchanger chromatography
(step 2)
'the immunoreactive Pool (about 200 mg) was further purified over a semi-
prepara-
tive reversed-phase (RP) chromatography at 45° C (Figure 3). The
immunoreactivily in
the hPTH-(53-84) -RIA ((Figure 3) and in the hPTH-(44-68)-RIA (Figure 3),
respectively, appeared in two peaks which were incompletely separated from
each
other.
Translation from PCT/EP90/U1807
~~~ ~.~v
- 13 -
The workup of the rear Pool which was higher reactive in the RIA is described
in
greater detail hereinbelow (Figures 4 to 6); after passage through the steps 4
to G il
resulted in the fraction No. 51.5 which was subjected to sequence analysis.
The fractions without designations did not contain any measurable
immunoreactivity.
4. a) Semi-preparative canon-exchanger chromatography of the immuno-
reactive pool front the semi-preparative RP-crhomatography (step 3)
The rear immunoreactive pool from the preceding RP chromatography was chromato-
graphed on a semi-preparative canon exchanger column at room temperature (Fig.
4),
and the medium-regional and C-Ierminal PTH-immunorcactivity (no Figure)
appeared
in the fractions 32-33. These were pooled (hatched area) and further
processed. The
fractions without designations did not contain any measurable
immunoreactivity.
b) Analytical RP-chromatography of lhc immunorcactivc pool from 4a)
The immunoreactive pool obtained in the preceding step of semi-preparative
cation-
exchanger chromatography was separated on an analytical RP column at
45° C (Fig.
5). The rear immunoreactive pool showing higher immunoreactivity (hatched) was
further processed. The fractions without designations did not contain any
measurable
immunoreactivity.
c) Analytical ration-cxhangcr chromatography of the immunorcactivc
pool from 4b)
The immunoreaclive pool was chromatographed on an analytical ration-exchanger
column at room temperature (Fig. G). The immunorcactivity in the hP'TH-(44-G8)-
RIA is seen in Figure G (hatched); the measurement in tl7c hPTI-I-(53-84)-RIA
resulted in the same picture. The fractions without designations did not
contain any
measurable immunorcactivity. The fractions exhibiting the highest
immunorcactivity were
pooled (hatched area) and further processed.
5. Hydrophobic interaction chromatography of the immunorcactivc pool
from 4c)
The immunoreactive pool from 4c) was further separated by means of the hydro-
phobic interaction chromatography at room temperature (Fig. 7), and the immuno-
reactivity was measured in the hPTH-(44-G8)-RIA (Fig. 7). The fractions
without
designations did not contain any measurable immunoreactivity. The fractions
exhibiting
the highest immunoreactivity were pooled (hatched area) and subjected to the
final
purification.
Translation from PCT/EP90/01i;07
_ 14 _ ~0~~ ;~e
6. a) Pirst analytical RP chromatography
The immunoreactive pool from 5) was chromatographed on an RP column at
25° C
and the immunoreactivity was monitored by means of the hPTH-(44-68)-RIA (Fig.
8). The fractions shown by the hatched areas were re-chromatographed.
b) Second analytical RP chromatography
The immunoreactive pool from 6a) was once more passed through an RP column
(Fig. 9). Except for the temperature which in this chromatography was 45
° C, the
conditions were the same as in step 6a). The material of the higher peak
designated
by the Code No. 51.5 was sequenced.
III. Isolation of a second fraction (Code No. 54.5) exhibiting immunoreacti-
vily in medium-regional- and C-terminal-specific radioimmunoassays for
parathormone (PTH)
During the isolation of the fraction 51.5, in the semi-preparative RP
chromatography
(step 3) there were obtained two pools exhibiting immunoreactivity. The work-
up of
the front pool of step 3 was carried out in a manner identical with that
described
for the rear pool in 11. 4 l0 6. In step 4b) again there were seen hvo immuno-
reactive peaks; in this case the front peak was predominant and was further
process-
ed. After passing the steps 4c) through 6c), a fraction designated by the Code
No.
54.5 was obtained, which was also subjected to sequence analysis.
IV. Sequencing the PTH peptides isolated from hcmofiltrale and exhibiting
immunoreactivity in medium- regional- and C-terminal--specific radioimmuno-
assays for parathormonc (PTH)
The fractions 51.5 and 54.5, the isolation of which from hcmofiltrate has been
described in II and III, were sequenced by means of the Gas Phase Sequcnator
Type
47(1 A (Applied Biosystems). The EDIvIAN degradation (Edma» a»d 13e~,~, Eur.
J. Bio-
chem. 1, 80-91, 1967) was carried out using the standard program PTHRUN. The
analysis of the derivatized amino acids was performed by the on-line method
using
an ABI 120 A chromatograph employing the standard protocol ABI.
For the fraction 51.5 a sequence of 39 amino acids could be determined; 5 more
amino acid moieties could be determined only ~~ith reservation. The sequence
found
conforms to the section (38-76) of human pTH (Ke»tmo»rr et al., Bioche»r. 17,
5723-
5729, 1978).
For the fraction 54.5 a sequence of 16 amino acids could be determined. The
sequence found conforms to human PTH-(38-53).
Translation from PCT/EP90/01807
2~~~ ~~~
- 15 -
Tu'o fractions could be isolated from human hcmofiltralc, both of which were
detected in a medium-regional as well as in a C-terminal PTH-RIA. Both
peptides
begin with the amino-terminal sequence Gly-Ala-Pro-Leu-Ala-Pro-Arg-etc., i.e.
both
peptides begin with the amino acid 38 of the hPTH molecule. For both of the
peptides found the last C-terminal amino acid cannot be indicated with
certainty. In
the case of the fraction 51.5, sequencing could be continued almost to the
position
84. The other peptide, of which only 16 positions could be determined by
sequence
analysis because of the smaller quantity available, has a sequence length of
at least
30 amino acids, because otherwise it would not be detected in the C-terminal
1RIA.
C-terminal PTH peptides having a different N-terminal beginning of the
sequence
could not be isolated. These results allow the conclusion to be drawn that the
hPTH-
(1-84) is cleaved between the amino acids 37 and 38 and that thereupon the
biologically active fragment hPTH-(1-37) is formed.
EXAMPLE 2
Synthesis of Human Paralhormone Peptide hPTH-(1-37)
1. Synthesis of the kicselguhr-reinforced Fmoc-Lcu-resin
'the Fmoc-amino acid anhydride (5 equivalents) was dissolved in a minimum
volume
of N,N-dimethylformamide (DMF) and was added to the hydroxymethylphenoxyacetyl-
norleucin resin in a round-bottom flask. 4-Dimethylaminopyridinc (1
equivalent) was
also dissolved in a minimum volume of DMF and added into the round-bottom
flask.
After one hour of reaction time the excess of the reagents was removed by
filtration,
and the resin was thoroughly washed on a filter.
2. Strategy of the synthesis of the human parathormone peptide
hPTI-I-(1-37)
For the synthesis of the peptide having the formula
Scr - Val - Ser - Glu - llc - Gln - Lcu - Met - 1-tis - Asn - Lcu - G1y - L>'s
- His - Lc;u - Asn - Scr - Met - Glu - Arg - Val - C~lu - '1'rp - Lcu - Arg -
Lys - Lys - Leu - Gln - Asp - Vat - His - Asn - I'hc - Val - Ala - Leu
the flow method (Atherton and Sheppard, Solid phase peptide n'ruhesis. IRL
Press, Oxford
1989) v~'as employed. The above-defined peptide sequence was synthesized by
means
of an automatic peptide synthesis apparatus (Milligen JO50) using the Fmoc-
penta-
fluorophenyl esters (OPfp). The follov~'ing Fmoc-amino acid OPfp's were used
in an
excess (4 equivalents) (in each case the derivatives of the L-amino acids were
employ-
ed).
Translation from PCT/EP~O/01807
CA 02071538 1999-11-02
- 16 -
Fmoc-Ala-OPfp Fmoc-Ala
Fmoc-Asp(OBut)-OPfp Fmoc-Asp(OBut)
Fmoc-Met-OPfp Fmoc-Met
Fmoc-Glu(OBut)-OPfp Fmoc-Glu(OBut)
Fmoc-His(Trt)-OPfp Fmoc-His(Trt)
Fmoc-Leu-OPfp Fmoc-Leu
Fmoc-Arg(Mtr)-OPfp Fmoc-Arg(Mtr)
Fmoc-Trp-OPfp Fmoc-Trp
Fmoc-Lys(BOC)-OPfp Fmoc-Lys(BOC)
Fmoc-Ile-OPfp Fmoc-Ile
Fmoc-Phe-OPfp Fmoc-Phe
Fmoc-Gly-OPfp Fmoc-Gly
Fmoc-Asn-OPfp Fmoc-Asn(Trt)
Fmoc-Gln-OPfp Fmoc-Gln(Trt)
Fmoc-Val-OPfp Fmoc-Val
Fmoc-Ser(But)-ODhbt Fmoc-Ser(But)
The synthesis can also be carried out by means of hydroxybenzotriazole esters
formed
in situ by the addition of TBTP (O-(1H-benzotriazol-1-yl)-N,N,N',N'-
tetramethyl-
uronium tetrafluoroborate~.
The peptide was removed from the carrier resin by addition of a mixture of
trifluoro-
acetic acid-anisole-ethanedithiol-phenol 94:2:2:2 (v/v/v/w) and precipitated
with ether.
The HPLC-purified peptide was characterized by amino acid analysis, analytical
HPLC
and amino acid sequence analysis.
3. Practical realization of the synthesis of the humane parathormone
peptide hPTH-(1-37)
The synthesis of
Ser - Val - Ser - Glu - Ile - Gln - Leu - Met - His - Asn - Leu - Gly - Lys
- His - Leu - Asn - Ser - Met - Glu - Arg - Val - Glu - Trp - Leu - Arg -
Lys - Lys - Leu - Gln - Asp - Val - His - Asn - Phe - Val - Ala - Leu (I)
hPTH-(1-37)
was carried out according to the continuous flow-method using the automatic
9050
PepSynthesizer* (Program Version 1.3) MilliGen/Biosearch. Fmoc-amino acid
penta-
fluorophenyl esters were used in the L-configurations and in portions of 0.8
mmoles
each. All reagents necessary for the synthesis were supplied from
Milligen/Biosearch:
N,N-dimethylformamide, 20% piperidine in N,N-dimethylformamide and 1-hydroxy-
benzotriazole. The L-amino acid derivatives were employed in a fourfold
excess. The
terminal amino groups were Fmoc-protected. Aspartic acid and glutamic acid
were
employed as N~'-tert-butyl esters; tyrosine and serine were employed as tert-
butyl
ethers; histidine and lysine were employed as NW-Boc compounds, and Arg was
*PepSynthesizer is a Trace-mark.
CA 02071538 1999-11-02
. - 17 -
employed as NG-2,2,5,7,8-pentamethylchromane-6-sulfonyl(Pmc) derivative. The
syn-
thesis was carried out starting from the kieselguhr resin Fmoc-Leu-Pepsyn KA
(MilliGen/Biosearch), that is with carrier-bonded C-terminal amino acid (0.091
meq
of leucine per gram, 1.60 g). The acylation of Fmoc-Arg(Pmc)-OH was carried
out
in the presence of ~O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
tetra-
fluoroborate~ (TBTU), 1-hydroxybenzotriazole and diisopropylethylamine. The
following
synthesis cycle was employed. Fmoc-removal with 20% of piperidine in DMF (7
min),
washing with DMF (12 min), acylation (30 min; for Fmoc-ValOPfp 45 min), and
washing with DMF (8 min). The progress of the synthesis was monitored by
continuous UV detection. The synthesis was terminated with the removal of the
N-
terminal Fmoc-group. The resin-bonded peptide was washed three times with 50
ml
each of isopropanol, glacial acetic acid, isopropanol and diethylether and
dried.
The removal from the carrier resin was effected with trifluoroacetic
acid/phenol/-
ethanedithiol/thioanisole/water 10:0.75:0.25:0.5:0.5 (v/w/v/v/v; 5 ml). The
solution is
concentrated in vacuo, and the product is precipitated by the addition of
diethylether.
The resulting crude peptide is washed several times with diethylether and
dried.
Thus, 65 mg of crude peptide are obtained from 240 mg of peptide resin.
The purification was carried out using a standard of hPTH-(1-37) prepared in
an
independent synthesis, the correct sequence of which had been confirmed by MS
(mass spectrometry) and sequence analysis. In the first step, the de-blocked
crude
material was purified by canon-exchanger HPLC (Parcosil PepKat, 2 cm x 10 cm,
300 A, 7 u., flow rate 9 ml/min, 230 nm, eluant A = 5 mM NaH2P04; B =
mM NaH2P04 + 1 M NaCI, gradient: 5% -~ 100% in 60 minutes), the main
peak appearing at 28.17 min (Figure 14) in concordance with the referene
substance.
In the second purification step, with simultaneous RP-HPLC for de-salting
(Parcosil
ProRP C4, 2 cm x 10 cm, 300 A, 7 u., flow rate 7 ml/min, 230 nm, eluant A =
0.1% trifluoroacetic acid in water; B = 0.1% trifluoroacetic acid in
acetonitrile/water
4:1, gradient 0% --j 100% B in 60 minutes), yield: 6.6 mg (1.5 mmoles; 14.8%),
there was obtained a peak at 30.80 min (Figure 15) which, in an analytical RP-
HPLC using C18 material gives a sharp uniform peak (Figure 16).
The identity of the synthesized material with the given primary structure of
PTH-(1-
37) was proven by MS (plasma desorption method, Bio-Ion, Applied Biosystems)
(Figure 17) and counter-sequencing in a Gas Phase Sequenator (Model 470,
Applied
Biosystems). The biological activity of the synthetic PTH-(1-37) was furnished
evidence of in the function test by differential muscle contraction of
pulmonal arteries
over Arteria renalis: The test showed that the synthetic material has the
correct biologi-
cal activity.
*Seq»enator is a Trademark.
- 1~
EXAMPLE 3
Evidence of the Biologically Active Circulating Human P'TH as hPTH-(1-37)
As described in Example 1, the hemofiltrate was worked-up by chromatographic
procedures, the synthetic hPTH-(1-37) according to Example 2 being utilized as
the
reference substance (Figure 10). Thus, from the hemofiltrate a fraction could
be
determined which exhibited immunoreactivity of amino-regional specificity in
the hPTA-
radioimmunoassay. This fraction was purified in the same manner as in Example
1
and characterized as hPTH-(1-37). Evidence was furnished of that by means of
the
Parcosil-RP column employed the hPTH-(1-37) can be unambiguously distinguished
from further PTH fragments (Figure 10), namely hPTH-(1-33), hPTH-(1-34), and
hPTH-(1-38).
EXAMPLE 4
Functional Analysis of hPTH-(1-37) for Furnishing Evidence of its Biological
Activity
Change of the intracellular level of cyclic 3',5'-adenosine monophosphate
(CAMP) in
phaeochromocytoma cells (PC-12) of the rat after application of humane parat-
hormone peptides hPTH-(1-33), hPTH-(1-37) and hPTH-(1-38).
In preliminary experiments it was shown that a phacochromocytoma line of the
rat
(PC-12) responds to an addition of PTH by increasing the intracellular CAMP
level.
Thereupon, the activity of the synthetic hPTH-(1-37) peptide was compared to
the
activities of other N-terminal PTH fragments (hPTH-(1-33) and hPTH-(1-38)~ in
this cell system.
A PC-12 cell line was used. The cells were cultured in RPMI medium 1 G4U
(Gibco)
with 10 ml/1 of L-glulamine 200 M (Gibco), lU°!o of equine scrum, 5% of
FCS, and
1% of penicillin/streptomycin. As the phosphadicslerase inhibitor there was
used 4-
isobutyl-1-methylxanthine (IBMX) from Sigma. Culture dishes comprising 24
wells
(1G mm in diameter) (Costar) coated with poly-L-lysine were used. Coating way
effected by applying a sterile-filtered poly-L-lysine solution (l0U mg/1) at
37° C for
one hour.
The following hPTH peptides were investigated:
hPT'H-(1-33) (MW 3971.2; peptide content 7G.9%); hPTH-(1-37) (MW 4401.0;
peptide content 74.5%); and hPTH-(1-38) (MW 4458.0; peptide content 81.8%).
The cells were used after the formation of a dense tell sheet (after about 4-8
days).
The incubation was carried out with 1 ml of cell medium at room temperature.
The
PTH peptides were diluted with cell medium immediately before the begin of the
assay. In the first run of the assay, 1.5 x 105 cells were incubated with
10~~M of
Translation from PCT/EP90/01807
CA 02071538 1999-11-02
- 19 -
hPTH-(1-33), hPTH-(1-37) and hPTH-(1-38) in the presence of 10-4M IBMX for
0, 2.5, 5, 10 and 20 minutes. For control, cells were incubated with only IBMX
for
20 minutes. The values are average values from triple runs ~ S.D. (cf. Fig.
11).
In a second experimental run, 2.2 x 105 PC-12 cells were incubated each with
10-11
(double run), 10'1, 10'9, 10'8 and 10-7M of each of hPTH- (1-33), hPTH-(1-37),
and hPTH-(1-38) in triple experiments in the presence of 10'~M IBMX for 5
minutes, and the intracellular cAMP concentration was measured. The IBMX
control
values without PTH were 18.0, 26.1, 20.9. The values depicted are average
values
S.D. (cf. .Figure 12).
Both PTH and IBMX were added at the same time. After suction-removal of the
medium the reaction was stopped by the addition of 1 ml of 99% ethanol. The
cells
were scraped off and were transferred into plastics tubes (Greiner) with the
ethanol.
The culture dish was rinsed with 66% ethanol, the supernatants were combined
and
centrifuged. The supernatant was evaporated on the water bath (50° C)
while purged
with nitrogen gas. The concentration of the cAMP was determined using a
radioimmunoassay kit (NEN). The measurement was carried out in accordance with
the producer's instructions. The amounts employed of cAMP standard, cAMP anti-
serum complex, cAMP 125I-tracer concentrate, c.~.'vfP carrier serum and cAMP
precipitator were ~0% of the amounts indicated by the producer.
The results show that significant differences exist in the activity of hPTH-(1-
37) in
comparison to the activities of other, non-endogenous PTH peptides in the cell
system investigated. This result allows to conclude differences in the
conformations of
these peptides which, as has been shown here, affect the activation of the PTH
receptor-adenylate cyclase system, but, moreover, also affect the
antigenicity.
EXAMPLE 5
Investigations Relating to the Secondary Structure of the Parathormone
Fragments
hPTH-(1-33), hPTH-(1-37) and hPTH-(1-37) by Circular Dichroism
The circular dichroism was measured by means of a Jasco J-500 Automatic
Recording Spectropolarimeter coupled with a Jasco DP-500 Data Processor. For
the
determination of the secondary structure, the measurement was carried out in
the
spectral range of from 190 nm to 240 nm at room temperature in a selected
quartz
cuvette at a light path of 1 mm. The parathormone concentrations employed were
50 l.~.g/ml of hPTH-(1-33), 50 l.tg/ml of hPTH-(1-37) and 60 ~tg/ml of hPTH-(1-
38). 10 mM Tris-HCI buffer pH 7.5 was used for dissolution (other conditions
of
the measurement: Sensitivity 2 milli-degrees/cm; time constant 2 seconds;
recorder
speed: 4 nm/min; wave length expansion: 5 nm/cm). The curves in Figure 13 for
hPTH-(1-33) hPTH-(1-37) and hPTH-(1-38) are average values for the signals
from
4 subsequent measurements minus the base line values. (,J~ is a Trade-park. )
.- 2 0 -
The evaluation for determining the secondary structure was performed in
accordance
with the method described by Reed and Kin~eJ (Biochernislry _23, 1357-1362,
1984).
From the data available, for the secondary structure of hPTH-(1-38) an cx-
helix pro-
portion of about 27% was calculated. The YTH fragment shorter by one amino
acid
[hPTH-(1-37)], in contrast thereto, has a significantly higher proportion of
the oc-
helix structure of about 40-45%. The amount of a-helix in the hPTH-(1-33) in
turn
is similarly low as in hPTH-(1-38).
From the investigation it ensues that, due to Lhe significant differences in
the
secondary structures, the peptide-receptor interactions and immuno-epitope
properties
of the PTH fragment hPTH-(1-37) must be clearly different from those of the
other
fragments.
EXAMPLE 6
lnvestigations Relating to the Biological Activity of hPTH-(1-37) onto the
Unstriated Muscles of the Pulmonary Vessels
and of the Corpm cavernosurn (Male Genital Organ)
The unstriated muscles of the lung vessels and of the Corpus cavernosum were
recovered from human surgical preparations and from rabbits and were fixed in
a
suitable organ bath system to measure the development of force. Il was
determined
that these muscles, in comparison to other smooth muscles of the human
organism,
after pre-contraction with various contraction-activating substances, exhibit
a distinct
relaxation after the addition of hPTI-I-(1-37) in concentrations in the
r<~ngc: of
10-9 moles, which furnishes evidence of the fact that the hPTH-(1-37) is a
particularly suitable peptide for the vasorclaxation of the pulmonary vessels
and the
activation of blood circulation of the male member.
Translation from PCT/EP9U/01807
