STABILIZED PTH FORMULATION
FIELD OF INVENTION
The invention provides aqueous stable pharmaceutical formulations comprising human
parathyroid hormone.
BACKGROUND OF INVENTION
Parathyroid hormone (PTH) is secreted by the chief cells of the parathyroid glands. These
glands are also involved in controlling the calcium amount in the blood and bones. They
are sensitive to small changes in Ca+2 concentrations. Initially, the parathyroid hormone is
synthesized as a larger preprohormone which is 115 amino acids in length. This
preprohomone is later cleaved in rough endoplasmic reticulum and then in Golgi
apparatus to form a biologically active hormone, which is an 84 amino acid peptide and
the molecular weight is 9425 daltons (Kim et al. 2009Korean J. Lab. Med. 29, 104-109).
The main biological active part of the PTH is the initial 34 amino-terminal amino acids.
The carboxyl terminal fragment of the PTH is biologically inactive. Further cleavage of
the PTH can occur either in the parathyroid glands or in the blood circulation. The
truncated PTH, which is produced by the cleavage from one or both (amino and carboxy)
terminal(s) has less or no biological activity.The secretion of PTH is controlled by a
negative feedback system. The circulating concentration of Ca+2 is detected by a unique
G-protein-linked calcium receptor (CaR). When the Ca+2 concentration increases, it
stimulates phospholipase C (PLC) and inhibits adenylatedcyclase (AC) which further
reduces PTH release and vice versa. It can be concluded that the PTH secretion is
inversely proportional to serum Ca+2 concentrations. When the Ca+2 concentrations are
within the normal limits, both the pathways are balanced and basal secretions of PTH are
maintained.
PTH acts on bones to increase the movement of Ca+2 from bone to blood. It also
stimulates osteocytes for bone formation as well as resorption. It enhances reabsoprtion of
Ca+ in the nephrons, reducing the excretion of Ca+ and stimulates calcitriol production
which increases intestinal absorption of Ca+2. In recent studies, for treating osteoporosis,
small amount of PTH is injected which helps in bone formation and bone strengthening
(Cosman et al. 2002 Osteoporos Int. 13(4), 267-77). PTH increases osteoblast production
rate and inhibits its apoptosis which further lead to an increase in skeletal mass and
improves bone micro-architecture (Lyritis et al. 2010 Ann. N. Y. Acad. Sci. 1205, 277-
283).
PTH is used as an anabolic agent for the treatment of osteoporosis (Black et al. 2003 N
Engl J Med. 349, 1207-1215; Jodar-Gimeno 2007 Clinlnterv Aging 2, 163-174;
Hodsman et al. 2003 ClinEndocrinolMetab. 88, 5212-5220). Two forms of recombinant
human parathyroid hormone (r-hPTH) are widely used. First form is hPTH(l-34) which is
34 residue amino-terminal of parathyroid hormone.hPTH (l-34)has a molecular weight of
4117.8 daltons and its amino acid sequence is shown as: H-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-OH. Recombinant parathyroid hormone highly
stimulates the bone formation than resorption (Resimini et al. 2011 Aging ClinExp Res
23, 30-32; Borba et al 2010 Arq Bras EndocrinolMetabol 54(2), 213-9). The second form
of r-hPTH is Preotact, which is the intact active 84 amino acid human PTH (1-84).
Proteins achieved through recombinant DNA technology are in a pure form. They are not
very stable under normal atmospheric conditions. So it becomes important to make a
stable pharmaceutical formulations which delays the degradation of the active principle
ingredient (API).Commercial usage of this hormone requires the development of a
formulation that will impart storage stability, retains the bioactivity and is easy to prepare.
Formulation of parathyroid hormone is labile due to degradation. It is more labile than the
traditional small molecules. It is highly sensitive to oxidation at methionine residues in
the positions 8 and 18 giving rise to oxidized PTH species. Furthermore it can get
deamidated at asparagine residue in position 16. There is a probability of truncation of
polypeptide chain at N-terminal and C-terminals due to breakage of peptide bond. All
these reactions can significantly hamper the bioactivity of this protein. Appropriate
formulation of PTH will prevent these adverse reactions.
US Patent Nos US 7,550,434; US 7,144,861 and US 6,770,623 discloses pharmaceutical
formulations comprising hPTH (1-34).
PCT application WO 2006/129995 discloses a liquid parathyroid hormone comprising a
parathyroid hormone.
SUMMARY OF THE INVENTION
In an embodiment, the invention is related to stable pharmaceutical formulations
comprising a biologically active hPTH and a buffer selected from Lactate buffer or
glutamate buffer.
In another embodiment, the invention is related to stable pharmaceutical formulation
comprisinghPTH and a buffer selected from lactate or glutamate having a pH range of 3.0
to 7.0.
In yet another embodiment, the invention is related to the pharmaceutical formulation
further comprising one or more tonicity agents or preservative.
In another embodiment, the parathyroid hormone is selected from the group consisting of
hPTH(l-34), hPTH(l-37), hPTH(l-38),hPTH (1-41) and hPTH (1-84).
In an embodiment, the invention is related to stable pharmaceutical formulation
comprising a biologically active hPTH (1-34) and a buffer selected from Lactate buffer or
glutamate buffer.
The details of one or more embodiments of the invention set forth in the below are
illustrative in nature only and not intended to limit to the scope of the invention. Other
features, objects and advantages of the inventions will be apparent from the description
and claims.
The present invention is related to a stable aqueous pharmaceutical formulation in a prefilled
syringe, vial, cartridge, or pen. In a preferred embodiment invention is related to a
stable aqueous pharmaceutical formulation in a vial, cartridge, or pen.
DETAIL DESCRIPTION OF INVENTION
The invention provides a stable aqueous pharmaceutical formulation comprising hPTH.
The pharmaceutical formulationsolution is sterile and can be stored for a long period of
time. The invention provides for a pharmaceutical formulation in a cartridge comprising a
stable hPTH and a buffer selected from lactate or glutamate. The formulation of the
invention is sterile and ready for parenteral administration.
In an embodiment of the invention, the biologically active hPTH is selected from the
group comprisinghPTH (1-34), hPTH(l-37), hPTH(l-38), hPTH (1-41) and hPTH (1-84).
The concentration of the hPTHisfrom lC^g/ml to 1000 g/ml, the preferred concentration
is 25 g/ml.
In an embodiment of the invention, lactic acid and sodium lactate constitute lactate buffer
and glutamic acid and sodium glutamate constitute glutamate buffer. In an embodiment of
the invention, the concentration of the buffer in the solution is ImM to lOOmM and the
preferred concentration is 10 mM.
In an embodiment of the invention, the buffering system used is acid and salt
combination, which is used to maintain the pH of the aqueous solution. In an embodiment
the pH range of the formulation of the invention is in the range of 3.0 to 7.0. The
preferred pH is 4.0.
In an embodiment of the invention, the stabilizing agentincorporated in the solution is
selected from a group of saccharide such as mannitol, glycine, glycerol ;chelators selected
from the group of EDTA, DTPA or EGTA; amino acid selected from the group of
proline, alanine, arginine, asparagines, aspartic acid, cysteine, glutamine, glutamic acid,
glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine,
threonine, tryptophan, tyrosine, and valine ;NaCland the like. The preferred stabilizing
agent ismannitol. The concentration of the stabilizing agent varies from about 2 to 20 wt-
% of the total solution.
In another embodiment of the invention, the stabilized aqueous composition comprises a
parenterally acceptable preservative. Examples of the preservatives are selected from a
group of cresols such as metacresol, paracresol, orthocresol; phenol, benzyl alcohol,
paraben, thimerosal, benzalkonium chloride, chlorobutanol, benzethonium
chloride,chlorobutanol and the like. The preferred preservative is metacresol and the
concentration range was about 0.1 to 2 wt of the total solution.
In another embodiment of the invention, the formulation is a stable hPTH (l-34)solution
comprising lactate or glutamate buffer, mannitol as a stabilizing agent and metacresol as a
preservative with a long shelf life at temperature ranging from 5°C to 40°C, preferably
5°C. The formulation has a long shelf life at 5°C.
In yet another embodiment, the invention is related to the method of treating a disease
using the stable pharmaceutical formulation of the present invention. The disease may be
glucocorticoid-induced osteoporosis in men and women or postmenopausal induced
osteoporosis in women or increase in bone mass in men with primary or hypogonadal
osteoporosis.
EXPERIMENTAL SECTION
The examples which follow are illustrative of the invention and are not intended to
belimiting.
Example 1
0.25 mg hPTH(l-34), 45.4 mg mannitol, 0.3 mg m-cresol, lOmM of lactic acid and
sodium lactate were mixed into a solution with 1 ml of distilled water. pH of the solution
was adjusted to 4.0 with sodium hydroxide or hydrochloric acid.
Table 1: Unit formula for the formulation of hPTH(l-34) of Example 1.
Components Formulation Unit composition Range
API hPTH(l-34) 0.25 mg 25-1000
Buffer Lactate Buffer l OmM 10-100 mM
Tonicity agent Mannitol 45.4 mg 2-20 wt %
Preservative Metacresol 0.3 mg 0.1-2 wt
PH 4.0 3-7
The buffer in this formulation is lactate buffer along with mannitol as a tonicity agent and
metacresol as a preservative. According to the results of RP-HPLC and SE-HPLC, it was
concluded that the Formulation of example 1 was stable.
Example 2
0.25 mg hPTH(l-34), 45.4 mg mannitol, 0.3 mg m-cresol, lOmM of glutamic acid and
sodium glutamate were mixed into a solution with 1 ml of distilled water. pH of the
solution was adjusted to 4.0 pH with sodium hydroxide or hydrochloric acid.
Table 2 :
The above formulations of Example 1 and Example 2 were prepared by gel filtration
chromatography (GFC) of drug substance which was in acetate buffer. GFC was carried
out for the buffer exchange to get the desired formulation where protein concentration
after buffer exchange was -0.6 mg/ml in respective formulation. It was further diluted
with the same buffer to achieve the final protein concentration of 0.25 mg/ml. These
formulations were filled aseptically into cartridges of volume 3 ml and were maintained
at 5°C and 40°C to check the stability of the protein. The stability of the protein at various
time points (0, 3,9 and 12months) was determined by checking protein profile by RPHPLC,
SE-HPLC. The pH, osmolality and bioactivity of hPTH(l-34) of the formulations
were determined after 12 months. Acetate estimation was carried out for all the
formulations to ensure appropriate buffer exchange during GFC step. The potency of
hPTH(l-34) was also calculated after 3 months and 12 months. Initially the potency of
example lwas 0.92 x 104 IU/mg, after 3 months the potency5°C was 1.27 x 104 IU/mg and
after 12 months the potency at 5°Cwas 1.14 x 104 IU/mg. Further, the potency of example
1 after 3 months at 40°C was 0.99 x 104 IU/mg. For example 2, the initial potency was 1.2
x 104 IU/mg, after 3 months the potency at 5°Cwas 0.85 x 104 IU/mg and after 12 months
the potency at 5°Cwas 1.21 x 104 IU/mg. Further the potency of example 2 after 3 months
at 40°C was 0.85 x 104 IU/mg.
The formulation of Example 1 and Example 2 were found to be stable at 5°C for a period
of more than one year.
The formulations of Example- 1 and Example-2 were found to be stable at 40°Cupto 4
weeks.
Example 3
Table 3:
Formulation
API hPTH(l-34)
Buffer Lactic acidSodium lactate
Tonicity agent Mannitol
Preservative Metacresol
pH 4.0
The formulation of this example comprisesmannitolas a stabilizing agent; lactic acid and
sodium lactate as buffering agents, which maintains the pH at 4.0 and metacresol as a
preservative. The concentration ofmannitol is 45.4 mg/ml.
Example 4
Table 4
The formulation of this example comprises glycerol as a stabilizing agent; lactic acid and
sodium lactate as buffering agents, which maintains the pH at 4.0 and metacresol as a
preservative. The concentration of glycerol is 23.02 mg/ml.
Example 5
Table 5
Formulation
API hPTH(l-34)
Buffer Lactic acid - Sodium lactate
Tonicity agent Glycine
Preservative Metacresol
PH 4.0
The formulation of this example comprises glycine as a stabilizing agent; lactic acid and
sodium lactate as buffering agents, which maintains the pH at 4.0 and metacresol as a
preservative. The concentration of glycine for this example is 18.76 mg/ml.
Example 6
Table 6
The formulation of this example comprises sodium chloride as a stabilizing agent; lactic
acid and sodium lactate as buffering agents, which maintains the pH at 4.0 and metacresol
as a preservative. The concentration of sodium chloride for this example is 7.3 mg/ml.
All patents, patent applications and publications cited in this application are hereby
incorporated by reference in their entirety for all purposes to the same extent as if each
individual patent, patent application or publication were so individually denoted.
Although certain embodiments and examples have been described in detail above, those
having ordinary skill in the art will clearly understand that many modifications are
possible in the embodiments and examples without departing from the teachings thereof.
CLAIMS
1. A stabilized aqueous pharmaceutical formulation comprising human parathyroid
hormone and a buffer selected from lactate or glutamate.
2. A stabilized aqueous pharmaceutical formulation comprising human parathyroid
hormone selected from the group of (1-34), (1-37), (1-38), (1-41), a buffer selected
from lactate or glutamate, a stabilizing agent and a parenterally acceptable
preservative, wherein the said formulation is sterile and ready for parenteral
administrationhaving pH in the range of 3 to 7.
3. The pharmaceutical formulation of claim 2, wherein the human parathyroid hormone
is hPTH (1-34).
4. The pharmaceutical formulation of claim 2, wherein the stabilizing agent is selected
from the group consisting of mannitol, glycine, glycerol, EDTA, DTPA or
EGTA,proline, alanine, arginine, asparagines, aspartic acid, cysteine, glutamine,
glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine,
phenylalanine, serine, threonine, tryptophan, tyrosine, valine and NaCl.
5. The pharmaceutical formulation of claim 4, wherein the stabilizing agent is mannitol.
6. The pharmaceutical formulation of claim 2, wherein the preservative is metacresol.
7. A stabilized aqueous pharmaceutical formulation comprising human parathyroid
hormone (1-34); a buffer selected from lactate or glutamate; mannitol as a stabilizing
agent and metacresol as a parenterally acceptable preservative; wherein the said
formulation is sterile and ready for parenteral administration.
8. The pharmaceutical formulation of claim 7, comprising about 10 g/ml to 1000
g/ml of hPTH (1-34), about ImM to lOOmM of lactate buffer,about 2 to 20 wt-% of
mannitol, about 0.1 to 2 wt% of metacresolhaving pH in the range of pH 3.0 to 7.0.
9. The pharmaceutical formulation of claim 7comprising aboutlO g/ml to 1000 g/ml
of hPTH (1-34), about ImM to lOOmM of glutamate buffer,about 2 to 20 wt-% of
mannitol, about 0.1 to 2 wt% of metacresolhaving pH in the range of pH 3.0 to 7.0.
10. A method for treating osteoporosis comprising administering the pharmaceutical
formulation of claim 1.