WO2001032201A2

WO2001032201A2 - A pharmaceutical composition having high cell membrane permeability

Info

Publication number: WO2001032201A2
Application number: PCT/US2000/026257
Authority: WO
Inventors: Michael Terrence Kuhfeld; Ovidio Vargas
Original assignee: Eli Lilly And Company
Priority date: 1999-10-29
Filing date: 2000-10-16
Publication date: 2001-05-10
Also published as: AU1189301A; WO2001032201A3; PE20010825A1

Abstract

A pharmaceutical composition useful for increasing the cell membrane permeability of PTH.

Description

A PHARMACEUTICAL COMPOSITION HAVING HIGH CELL MEMBRANE PERMEABILITY

Field of the Invention

The present invention relates to the fields of pharmaceutical and bio-organic chemistry and provides a pharmaceutical composition having high cell membrane permeability.

Background of the Invention

Numerous active ingredients suffer from the disadvantage of being poorly soluble in aqueous medium, such as that in the physiological system i.e., the gastrointestinal tract of a mammal. This results in an insufficient dissolution profile, and consequently poor bio-availability within the organism following oral administration, transdermal administration or other application of the pharmaceutical agent. This often results in a necessity to increase the therapeutic dose required to obtain or maintain desired intracellular or serum concentration of the pharmaceutical agent. Aungst has surmised that poor oral bio-availability and attendant pre-systemic degradation is typically encountered in the efforts to develop oral proteins, peptides and peptidomimetic agents (Aungst B. J.; Journal of Pharmaceutical Sciences vol. 82, No. 10, 1993).

Certain peptides and proteins are known to degrade due to the acidic pH of the gastrointestinal tract or due to metabolism by digestive enzymes or luminal metabolism. The propensity for peptides and proteins to be digested is aided by the slow permeability through the membranes, a factor attributable to the large size of the proteins and their lipophilic folding patterns (Samanen, J. J. Pharmaceutical Pharmacology, 48, 119-135, 1996) .

It has long been recognized that the absorption, hence bio-availability of proteins can be enhanced by means including use of lipid vehicles or surfactant vehicles to aid in transport through the membrane (Constantinides , R. P., Pharmaceutical Research, vol. 12, No. 11, 1561-1572, 1995) . However, because of the unique characteristic of one protein compared to another or the characteristics of a protein in different environments, the workable methods for improving bio-availability are substrate specific and often reagent specific. Thus, it is not predictable that a particular surfactant would enhance the absorption of a specific protein. Where enhancement has been observed the degree of enhancement is also substrate specific and unpredictable. It is also known that certain transport mechanisms result in damage to the cell membrane (i.e., cell membrane lysis), making it even more elusive to find pharmaceutically suitable agents that enhance the bio-availability of particular protein substrates without deleterious effects on the cell membranes.

In general surfactants used in the enhancement of bio-availability have the drawback of non-reversibility. Thus, often the cell membrane is irreversibly disrupted as a result of contact with the surfactant.

Formulations that use surfactants have typically used these agents as wetting agents that help to maintain the integrity of the formulation. Wetting improves such factors as stability, cohesiveness and provision of hydrophilic sites for interaction with the cell membrane. Summary of the Invention

The present invention provides a novel composition for enhancing the bio-availability of PTH using a formulation containing a betaine zwitterionic surfactant,

In one embodiment of the invention is a pharmaceutical composition comprising PTH and lauramidopropyl betaine surfactant.

In one embodiment of the invention the use of the betaine zwitterionic surfactant lauramidopropyl betaine for the enhancement of the cell membrane permeability of PTH is provided, particularly the use of lauramidopropyl betaine in combination with the 1-34 a ino acid fragment of PTH.

In another embodiment of the invention an improved method of treating bone disease is provided which comprises the administration of a pharmaceutical composition (s) comprising PTH and lauramidopropyl betaine .

In yet another embodiment of the invention the use of a pharmaceutical composition of PTH particularly, the 1-34 amino acid fragment and lauramidopropyl betaine for the treatment and/or prophylaxis of bone loss or bone thinning in mammals, including a human is provided.

The present invention includes the use of the zwitterionic surfactant lauramidopropyl betaine ( APB) , to improve the cell membrane permeability of PTH.

The present invention provides enhancement of the cell membrane permeability of PTH of human, bovine or recombinant origin or modifications thereof.

The present invention relates to the use of a pharmaceutical composition comprising PTH and lauramidopropyl betaine in therapy . The present invention provides a method for the enhancement of the cell membrane permeability of PTH, including the full length (1-84 aminoacid) and fragments thereof i.e. the 1-34 amino acid fragment by the use of a betaine zwitterionic surfactant.

The present invention is also a method for treating bone loss comprising administering a pharmaceutical composition of PTH, lauramidopropyl betaine zwitterionic acid surfactant and raloxifene™ as separate dose units either simultaneously or sequentially within a therapeutically effective interval.

The present invention is also the use of PTH, betaine zwitterionic acid surfactant and a co-agent in the manufacture of a medicament for the treatment and/or prophylaxis of bone loss.

Definitions

Excipients are inert substances such as, without limitation, carriers, diluents, flavoring agents, sweeteners, lubricants, solubilizers , suspending agents, binders, tablet disintegrating agents and encapsulating material . Suitable excipients include derivatives of sugars, such as lactose, saccharose, hydrolysed starch (malto-dextrin) , and the like. Mixtures are also suitable .

As used in the framework of the invention the expression "inert hydrosoluble carrier" is synonymous with "carrier" and means generally any hydrophilic, pharmaceutically inert, crystalline or amorphous, in a particular form, not leading to a chemical reaction under the operating conditions employed, and which is soluble in an aqueous medium, notably in a gastric acid medium. The carrier may serve as a diluent, which may be solid, semi-solid, or liquid material which acts as a vehicle, or can be in the form of tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments, containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions and sterile packaged powders. One of skill in the art is aware that the exact nature of the carrier is not critical provided it neither interacts negatively with PTH or PTH and co- agents nor negatively affect the function of the betaine surfactant ( s) utilized.

As used herein SR101 is used to represent sulforhodamine 101 hydrate or other sulforhodamine analog having fluorescent properties. Sulforhdamine hydrate and analogsd thereof, are available from Aldrich Chemical Company, Milwaukee USA, and other chemical suppliers. The exact fluorescent marker is not critical to the practice of the invention.

As used herein the term "TEER" is synonymous with the term "TER" also used in the literature and is an abbreviation for transepithelial electrical resistance. The TEER value is a measure of the electrical resistance to flow across a cell membrane, and is a measure of the cell membrane integrity.

The phrase " therapeutically effective interval" is a period of time beginning when one of either PTH or a betaine zwitterionic surfactant, i.e. lauramidopropyl betaine is administered to a mammal and ending at the limit of the therapeutic effectiveness of either PTH or the limit of permeability enhancement of lauramidopropyl betaine.

The term "zwitterionic surfactant" as used herein refers to its conventionally known meaning in this invention. Lauramidopropyl betaine is a member of the class of surfactants known as the betaines . It is a zwitterionic surfactant, which means that at a pH range of about 6.0-7.4, both positive and negative charges are present in the molecule. Betaines in general have found use as cleaning agents, and personal care products. However, their use as permeability enhancers for the enhancement of PTH absorption has not been realized. Lauramidopropyl betaine is represented by the formula:

and is an example of a betaine zwitterionic surfactant.

Detailed Description

Parathyroid hormone has been observed to be useful for the treatment of bone loss . Bone loss affects a significant population of adults over age 50, particularly, post menopausal women. The symptoms of bone loss include loss of posture or gait, decreased height, more frequent bone fractures among others.

The enhancement of PTH absorption is present regardless of form or source of PTH ^~i . e . , human, bovine or recombinant origin or the full length 1-84 amino acid sequence or the 1-34 amino acid fragment or other fragment or modification thereof. Particularly preferred for the purpose of the invention is a composition consisting of the 1-34 amino acid fragment from the N- terminus of PTH, and a betaine zwitterionic surfactant for.

The practice of the invention involves increasing the transport of PTH through the cell membrane in the presence of a betaine surfactant as an agent for increasing the bio-availability of PTH. The effect of a betaine surfactant i.e., lauramidopropyl betaine surfactant on the permeability of PTH through caco-2 cells was modeled in-vi ro. Modeling experiments were performed with the aid of an automated permeability machine. The automated permeability machine has been shown to effectively model the transport of molecules from outside the cell membrane into the intracellular space. The details of the permeability analysis machine are disclosed in US. Patent Application No. 09/068,315 filed January 11, 1999, the entire content of which is incorporated herein by reference. The method of modeling and the apparatus for modeling are not aspects of the present invention nor are they critical to the practice of the present invention. Cell permeability modeling experiments have been described in the literature including Artursson, P., and Karlsson, J., Biochem . Biophysics Research Communication , 175, 880-885, 1991; Hilgers et al . , Pharm . Research, 7, 902-910, 1990; Hu, M. and Borchardt, R. , Pharm . Research, 7, 1313-1319, 1990; and Hochman et al . , J. Pharmacology and Experimental Therapeutics , 269,(2), 813-822, 1994.

Results obtained indicate that lauramidopropyl betaine at concentrations of about 0.05% (w/w) of PTH or less effects a reversible interaction with the cell membrane or intercellular spaces and joints so that the integrity of the cell membrane is maintained after passage of PTH.

The improved cell membrane permeability of PTH achieved through the use of a betaine surfactant offers the advantage of improved bio-availability. The present invention relates to a novel pharmaceutical composition having high bio-availability through improved dissolution and permeability through cellular membranes. The betaine surfactants are especially preferred for the practice of the present invention. Suitable betaine zwitterionic surfactants include the propylbetaines selected from the group consisting of caproylamidopropyl betaine, cocodimethylpropyl betaine, cocamidopropyl betaine, oleoylamidopropyl betaine, lauramidopropyl betaine and palmitoylpropyl betaine. Particularly preferred is lauramidopropyl betaine.

The absorption of a PTH formulation (without a betaine surfactant) through human caco-2 cell membranes was compared with that of a PTH formulation containing a betaine surfactant through human caco-2 cell membranes . Human caco-2 cells have been known to form monolayers which mimic many of the morphological and absorptive properties of cells (Pinto et al . , Biol . Cell 47:323-330, 1983; Hidalgo et al . , Gastroenterology, 96, 736-749, 1989; Vanchon, P.H. and Beaulieu J.F., Gastroenterology, 103, 414-412, 1992). Caco-2 cells have been used extensively as a model to predict drug absorption across the intestinal epithelium, see Artursson, P., and Karlsson, J., Biochem . Biophysics Research Communication, 175, 880-885, 1991; Hilgers et al . , Pharm . Research, 7, 902-910, 1990; Hu, M. and Borchardt, R. , Pharm . Research, 7, 1313-1319, 1990. Caco-2 cells have also been used to evaluate the effect of absorption enhancers on drug transport, Hochman et al . , J. Pharmacology and Experimental Therapeutics , 269,(2), 813-822, 1994.

In a preferred embodiment of the invention, PTH is delivered after the cell membrane has been affected with lauramidopropyl betaine or similar betaine zwitterionic surfactant according to the invention. The simultaneous or premixed delivery of LAPB and PTH results in a smaller enhancement of PTH absorption through the cell membrane. Affecting the cell membrane with lauramidopropyl betaine may be accomplished by administering a unit dose composition of lauramidopropyl betaine to the recipient followed by administering a unit dose composition of PTH to the recipient within a therapeutically effective interval .

The enhancement in absorption of PTH through cell membrane in the presence of a zwitterionic surfactant, for example lauramidopropyl betaine, was evaluated using the following procedure. Test compounds were prepared using DMSO or other water miscible solvent such that the resulting test concentration was between 25 and 100 micromolar (μM) of PTH. Caco-2 cells, model intestinal epithelial cells derived from human colorectal carcinoma were grown on Millicell-PC™ (0.4μM pore size, 30mm diameter, Millipore Corporation, Bedford, Massachusetts, U.S.A. ) filters.

TEER measurements were performed as described above to check barrier integrity before the filters were loaded on to the diffusion chambers resulting in the chamber being divided into donor (apical) and receiver (basolateral) sides. Solutions or suspensions of the test compound for example, PTH along with the absorption promoter for example, lauramidopropyl betaine was added to the donor chamber. Typically four experiments were run simultaneously, with one experiment being the control. The control experiment is identical to the test experiments but for the lack of an absorption promoter, e.g. lauramidopropyl betaine. Test samples were taken from both the donor and receiver chambers over four hours and analyzed by HPLC or other generally acceptable analytical technique for the concentration of PTH. The enhancement factor calculated by calculating the percentage of PTH in the receiver chamber compared to the initial amount in the donor chamber and comparing this percentage to that obtained for a control sample where LAPB was not added.

Alternatively, the absorption of PTH in the presence of lauramidopropyl betaine may be measured by adding a paracellular leakage marker, such as for example SR101™, Lucifer Yellow or other fluorescent marker to the donor chamber containing PTH and lauramidopropyl betaine or other betaine zwitterionic surfactant, and analyzing samples obtained over time for the percentage of marker that had been transported to the receiver chamber. This is compared against the percentage of paracellular marker transported to the receiver chamber in a control sample where the leakage marker was added to a solution containing PTH but not lauramidopropyl betaine or other zwitterionic surfactant. Typically, SRI01™ for example, is added to the donor chamber and test samples of the marker are taken from both the donor and receiver chambers over time, typically over 10 minutes to 4 hours and preferably over 20 minutes to 30 minutes. The marker test sample concentrations were analyzed by fluorescence spectroscopy and a leakage percentage (concentration of marker in receiver chamber/concentration of marker in donor chamber of PTH in donor chamber *100) over time is calculated.

The reversibility of the effect of PTH absorption in the presence of lauramidopropyl betaine through cell membrane, on the cell membrane, was evaluated by analyzing the cell membrane's ability to maintain its integrity after lauramidopropyl betaine enhanced transport (absorption) of PTH. Typically, agents that enhance the transport of protein through the cell membrane and hence improve the bio-availability of drug candidates often effect irreversible destruction of the cell membrane, which is an undesirable effect. However, applicants have discovered that the use of lauramidopropyl betaine, for example, does not result in an irreversible effect on the cell membrane at concentrations of less than 0.05% (weight/weight ) , preferably at concentrations of less than 0.03% (w/w) of PTH.

Cell membrane integrity is determined by transepithelial electrical resistance (TEER) measurements. Typically the caco-2 cells (GI tract epithelial cell model) are cultured in a growth medium (GIBCO BRL™, DMEM/F-12 3:1, with 5%FBS, 1.0% HEPES and 0.1% tobramycin, GIBCO Laboratories, Grand Island, NY) over a three week period and impregnated on a polycarbonate filter.

Transepithelial electrical resistance (TEER) of the caco-2 impregnated filters is analyzed to provide a baseline (control) value. This is followed by addition of lauramidopropyl betaine to the caco-2 impregnated filter. TEER measurements are then taken periodically over a 1 to 4 hour period, preferably over a 1 to 2 hour period. The TEER measurements after LAPB addition were found to be lower than before LAPB addition. The LAPB was then washed off the filter. Fresh growth medium is impregnated on to the filter. TEER measurements taken from 4 to 17 hours after impregnation of filter with new growth medium showed a recovery of TEER values to near or about the level prior to LAPB addition.

Typically, four experiments are conducted which included a control (40mL of growth media in the TEER measurement device plus 4mL of growth media on the donor side of the filter) and three with varying percentages of surfactant preferably including, 4mL each of 0.01%, 0.03% and 0.05% lauramidopropyl betaine (LAPB) in 4mL of growth media respectively. TEER measurements are taken over a period of 1 hour. The three samples are emptied and the sample cells are cleaned. The test samples are reconstituted as above but without LAPB, and TEER measurements repeated over time for up to 17 hours . Applicants have found that the integrity of the cell membranes is maintained (from about 20-90% of initial value) over time and that the surfactant lauramidopropyl betaine did not irreversibly disrupt the cell membrane between the concentration levels of from about 0.01% to about 0.05%.

Experimental PTH may be synthetic or recombinant in origin and prepared by established procedures. PTH 1-34 may be purchased from Bachem Corporation of Torrance, California. LAPB was obtained from the Mclntyre Group Limited, University Park, Illinois.

Examples

Examples 1-4

Three diffusion chambers were prepared to study the effect of lauramidopropyl betaine (LAPB) on in-vi tro PTH permeability through caco-2 cells. Caco-2 cells were used to model in-vivo permeability in mammalian intestinal cell membranes. A 23:1 ratio of surfactant to PTH was tested. (15.6 mg/mL LAPB to 0.67 mg/mL PTH) in two chambers and a control value for PTH transport at 0.67 mg/mL was established in a third diffusion chamber devoid of LAPB.

CaCO-2 cells, cultured on filters according to standard procedures, were used as the in-vitro cell barrier. TEER measurements were taken to ensure barrier integrity before the transport experiment was run.

The donor chambers were preloaded with 15.6 mg/mL LAPB and the transport experiment was started with the addition of PTH to each of the three donor chambers.

Two donor (initial and final) and four receiver samplings taken during the course of a two-hour experiment were analyzed by HPLC . At the end of the transport experiment, paracellular leakage marker SR101 was added to measure barrier integrity of the caco-2 cell membranes. SR101 % transported values (leakage percentage) in excess of the established threshold value (0.2%) indicate an alteration of CaCO-2 cell membranes during the course of the transport experiment.

For the control chamber, a permeability value of less than 1 X 10^~^cm/min is consistent with previous PTH control values generated. In the other two chambers, PTH permeability in the presence of 20 to 1 excess LAPB was enhanced over 500-fold relative to control values See table 1 below) .

Table 1 Permeability Enhancements (Papp)

Description of % LAPB in Ratio of Papp

Donor donor LAPB to

(w . /vol . ) test compd 0.67mg/mL 0.0 1

PTH

0.39mg/mL PTH + 0.05 1.3 to 1 145

0.5mg/mL LAPB

0.39mg/mL PTH + 0.05 1.3 to 1 153

0.5mg/mL LAPB

0.67mg/mLPTH + 1.30 20 to 1 549

13.4mg/mL LAPB

0.67mg/mL PTH + 1.30 20 to 1 587

13.4mg/mL LAPB

Transport Experiment Barrier Integrity Results The results of SR101 percent transport values (leakage tests) are presented in table 2 below:

Table 2

Description of TEER % SR101

Donor transported

0.67mg/mL 690 0.06

PTH

0.39mg/mL PTH + 2286 3.01 0 . 5mg/mL LAPB

0 . 39mg/mL PTH + 772 3 . 26

0 . 5mg/mL LAPB

0 . 67mg/mL PTH + 1498 1 . 63

15 . 6mg /mL LAPB

0 . 67mg/mL PTH + 1927 1 . 73

15 . 6mg/mL LAPB

Examples 5-8

Three separate concentrations of LAPB (0.01%, 0.03%, 0,05%) were prepared in growth media (GM) (GIBCO BRL™, DMEM/F-12 3:1, with 5%FBS, 1.0% HEPES and 0.1% tobramycin, GIBCO Laboratories, Grand Island, NY) . A 40 mL sample of GM was placed in the TEER apparatus while a 4 mL sample of GM was placed on the apical side of each filter containing the CaCO-2 cells. The initial TEER values were read on all filters prior to the addition of LAPB while leaving one filter devoid of LAPB to be used as a control. A 4 mL sample of GM was removed from each of the other filters and replaced with 4 mL of GM containing LAPB at the appropriate concentration. TEER readings in the presence of LAPB were taken every 5 minutes for a total of 1 hour.

After 1 hour, LAPB was removed from the filters by washing with fresh GM and the filters were placed in the incubator (37 °C, 5% CO2 ) overnight . In the morning, TEER values were again taken to determine whether cell barrier integrity had been restored to those filters previously affected by LAPB. A table showing the results of reversibility experiments is presented below (table 3): Table 3 Results of Reversibility Experiments

Concentration of TEER TEER after TEER after

LAPB before LAPB washing off

LAPB exposure LAPB exposure

0.0 719 X 625

0.01 706 580 650

0.03 523 310 445

0.05 650 37 298 is control value with no LAPB at end of experiment

PTH and lauramidopropyl betaine may be administered sequentially as unit dose compositions to the subject, preferably in the order LAPB, PTH. Compositions containing PTH and LAPB may also be administered simultaneously as individual unit doses or in a premixed composition as a single unit dose. The period of administration of PTH will typically be one week to one year, and optimally, one week to six months.

Pharmaceutical formulations or compositions of the invention which include PTH and lauramidopropyl betaine for administration will generally include a pharmaceutically effective amount of the bone growth factor (PTH) to promote bone growth in addition to a pharmaceutically acceptable excipient. Suitable excipients include most inert hydrosoluble carriers approved for parenteral administration, including water, saline, Ringer's solution, Hank's solution, and solutions of glucose, lactose dextrose, ethanol, glycerol, albumin, and the like. These compositions may optionally include stabilizers, antioxidants, antimicrobials, preservatives, buffering agents, other surfactants, and other accessory additives. PTH and lauramidopropyl betaine may also be delivered in an ionophoretic patch. A thorough discussion of suitable vehicles for parenteral administration may be found in E.W. Martin, "Remington's Pharmaceutical Sciences" (Mack Pub. Co.). Such formulations are generally known to those skilled in the art and are administered systemically to provide systemic treatment .

The composition comprising PTH and lauramidopropyl betaine has the weight ratio of PTH to lauramidopropyl betaine will be from about 0.01:100 to 10:100, preferably from about 1:100 to about 5:100, and optimally about 3:100.

The precise dosage necessary will vary with the age, size, sex and condition of the subject, the nature and severity of the disorder to be treated and the like. Thus, a precise effective amount cannot be specified in advance and will be determined by the caregiver. However, appropriate amounts may be determined by routine experimentation with animal models. In general terms, an effective dose of PTH for systemic treatment will range from about 0.001 μg/kg to about 100 mg/kg of body weight, per day .

Suitable carriers also include hydrogels, controlled- or sustained-release devices (e.g., an Alzet ® minipump) , polylactic acid, and collagen matrices. Presently preferred carriers are formulations of atelopeptide collagen containing particulate calcium phosphate mineral components, such as combinations of homologous or xenographic fibrillar atelopeptide collagen (for example Zyderm® Collagen Implant, available from Collagen Corporation, Palo Alto, Calif.) with hydroxyapatitetricalcium phosphate (HA-TCP, available from Zimmer, Inc., Warsaw, Indiana).

In general, implant devices may be coated with a composition comprising PTH and lauramidopropyl betaine as follows. The PTH and lauramidopropyl betaine is dissolved at a concentration in the range of 0.01 μg/mL to 200 mg/mL in phosphate-buffered saline (PBS) solution containing 2 mg/mL serum albumin. The porous end of an implant is dipped in the solution and is air-dried (or lyophilized) or implanted immediately into the bony site. The viscosity of the coating solution is increased, if desired, by adding hyaluronate at a final concentration of 0.1 mg/mL to 100 mg/mL or by adding other pharmaceutically acceptable excipients. Alternatively, the solution containing PTH and lauramidopropyl betaine is mixed with collagen gel or human collagen (e.g. Zyderm® Collagen Implant, Collagen Corp., Palo Alto, Calif.) to a final collagen concentration of 2 mg/mL to 100 mg/mL to form a paste or gel, which is then used to coat the porous end of the implant device. The coated implant device is placed into the bony site immediately or is air-dried and rehydrated with PBS prior to implanting, with the objective of maximizing new bone formation into the implant while minimizing the in-growth of soft tissue into the implant site.

Preferably the pharmaceutical formulation is in unit dosage form. The dosage unit (unit dose) form can be a capsule or tablet itself, or the appropriate number of any of these. The quantity of PTH in a unit dose of composition may be varied or adjusted from about 0.1 to about 1000 milligrams or more with sufficient lauramidopropyl betaine to effect efficacious delivery according to the particular treatment involved. Unit dosage forms can also be individual units of PTH and individual units of lauramidopropyl betaine to be administered simultaneously or sequentially within a therapeutically effective interval.

Compositions (dosage forms) suitable for internal administration contain from about 1 milligram to about 500 milligrams of PTH. In these pharmaceutical compositions the PTH will ordinarily be present in an amount of about 10 to 90% by weight, lauramidopropyl betaine will ordinarily be present in an amount of about 0.5 to 10% by weight, based on the total weight of the composition.

Examples of useful pharmaceutical compositions and their proportions of ingredients are illustrated as follows :

Capsules: Capsules may be prepared by filling standard two-piece hard gelatin capsules each with 50 mg of PTH, 2.0 mg of lauramidopropyl betaine, 175 mg of lactose, 24 mg of talc, and 6 mg of magnesium stearate.

Soft Gelatin Capsules: A mixture of PTH and lauramidopropyl betaine in soybean oil is prepared and injected by means of a positive displacement pump into gelatin to form soft gelatin capsules containing 50 mg PTH and 2.0 mg of lauramidopropyl betaine. The capsules are washed in petroleum ether and dried. Tablets: Tablets may be prepared by conventional procedures so that the dosage unit is 50 mg of PTH, 2.0 mg of lauramidopropyl betaine, 6 mg of magnesium stearate, 70 mg of microcrystalline cellulose, 11 mg of cornstarch, and 225 mg of lactose. Appropriate coatings may be applied to increase palatability or delay absorption.

Suspensions: An aqueous suspension is prepared for oral administration so that each 5 mL contain 25 mg of PTH, 0.75 mg of lauramidopropyl betaine, 200 mg of sodium carboxymethyl cellulose, 5 mg of sodium benzoate, 1.0 g of sorbitol solution, U.S. P., and 0.025 mg of vanillin.

Injectables: A parenteral composition suitable for administration by injection is prepared by stirring 1.5% by weight of PTH and 0.075% by weight of lauramidopropyl betaine in 10% by volume propylene glycol and water. The solution is sterilized by commonly used techniques.

Nasal Spray: An aqueous solution is prepared such that each 1 mL contains 1 mg of PTH, 0.05 mg of lauramidopropyl betaine, 1.8 mg methylparaben, 0.2 mg propylparaben and 10 mg methylcellulose . The solution is dispensed into 1.0 mL vials.

The PTH may be used at a concentration of 0.1 to 90% weight percent of the formulation.

Aerosol formulations are capable of dispersing into particle sizes of from about 0.5 to about 10 microns and have sufficient PTH and lauramidopropyl betaine to achieve concentrations of PTH on the airway surfaces of from about 10¹⁰ to 10^"2 moles per liter. Transdermal patch: An aqueous solution is prepared such that each 1 mL contains 1 mg of PTH, 0.05 mg of lauramidopropyl betaine, 1.8 mg methylparaben, 0.2 mg propylparaben and 10 mg methylcellulose . Other excipients may be added to provide a non-runny, consistency suitable for application to a surface for use as a patch. In general, methods known to one of skill in the art including controlled delivery methods are contemplated as within the invention.

An aspect of the present invention is the use of a composition comprising PTH and lauramidopropyl betaine in the combination therapy of PTH and other therapeutically effective treatments or prophylactics for bone diseases, including bone loss. Co-agents for PTH useful for the purpose of the present invention include bisphosphonates, calcitonin, hormone replacement agents, fluorides and calcium supplement agents with or without vitamin D. The methods and compositions of the invention are useful for treating bone fractures, defects, and disorders which result in weakened bones such as osteoporosis, osteoarthritis, Paget ' s disease, osteohalisteresis, osteomalacia, bone loss resulting from multiple myeloma and other forms of cancer, bone loss resulting from side effects of other medical treatment (such as steroids) , and age-related loss of bone mass.

In accordance with one method of use, the composition of PTH and lauramidopropyl betaine may be administered systemically, orally and/or parenterally, including subcutaneous or intravenous injection, and/or intra-nasally. The compositions described above may be used in the manufacture of a medicament for the treatment and/or prevention of bone loss in a mammal, including a human.

Claims

We Claim :

1. A pharmaceutical composition comprising

(a) parathyroid hormone, and

(b) a betaine zwitterionic acid surfactant.

2. The pharmaceutical composition of Claim 1 wherein said betaine zwitterionic acid surfactant is a propyl betaine zwitterionic surfactant.

3. The pharmaceutical composition of Claim 1 wherein said betaine zwitterionic acid surfactant is lauramidopropyl betaine .

4. The pharmaceutical composition according to Claim 1 further comprising a carrier and/or excipient.

5. A pharmaceutical composition according to any one of Claims 1-4 wherein said betaine zwitterionic acid surfactant is in the weight ratio of PTH to betaine surfactant of from about 0.05 : 100 to about 10.0 : 100.

6. A pharmaceutical composition according to any one of Claims 1-5 wherein said lauramidopropyl betaine zwitterionic acid surfactant is in the weight ratio of PTH to lauramidopropyl betaine of from about 0.03 : 1 to about 1 : 23.

7. A method of treating bone loss comprising administration of a therapeutically effective amount of PTH and betaine zwitterionic acid surfactant.

8. The use of a therapeutically effective amount of PTH and betaine zwitterionic acid surfactant for treating and/or preventing bone loss.

9. The use of a therapeutically effective amount of PTH and lauramidopropyl betaine for treating and/or preventing bone loss.

10. A method of treating bone loss according to Claim 5 wherein the formulation of the composition is delivered parenterally .

11. A method of treating bone loss according to Claim 5 wherein the formulation of the composition is delivered as a transdermal patch.

12. A method of treating bone loss according to Claim 5 wherein the formulation of the composition is delivered orally.

13. A method of treating bone loss according to Claim 5 wherein the formulation of the composition is delivered by intra-nasal administration.

14. A method for the treatment or prophylaxis of an affliction in a mammal including a human requiring parathyroid hormone wherein the bio-availability of parathyroid hormone is enhanced by the use of a therapeutically effective amount of a composition comprising PTH and lauramidopropyl betaine.

15. An improved method for treating bone loss consisting of PTH and inert carriers and/or excipients wherein the improvement comprises administering to a mammal in need thereof a therapeutically effective amount of a composition comprising PTH and lauramidopropyl betaine

16. A method for treating bone loss comprising administration of PTH and lauramidopropyl betaine as single dose units or as separate dose units either simultaneously or sequentially within a therapeutically effective interval .

17. A method for treating bone loss according to Claim 13 further comprising administering PTH and lauramidopropyl betaine as separate dose units either simultaneously or sequentially within a therapeutically effective interval.

18. A method of treating bone loss further comprising administering PTH and lauramidopropyl betaine as separate dose units sequentially within a therapeutically effective interval.

19. A method for treating bone loss comprising administering a therapeutically effective amount of PTH and lauramidopropyl betaine as single dose units wherein a PTH composition is administered following administration of a lauramidopropyl betaine composition.

20. A pharmaceutical composition comprising PTH and lauramidopropyl betaine useful in the manufacture of a medicament for the treatment and/or prevention of bone loss in a mammal .

21. The use of a pharmaceutical composition comprising PTH and lauramidopropyl betaine in therapy.

22. The use of PTH and lauramidopropyl betaine in the manufacture of a medicament for the treatment or prophylaxis of an affliction in a mammal including a human requiring parathyroid hormone.

23. The use of a therapeutically effective amount of PTH and lauramidopropyl betaine for the manufacture of a medicament for the treatment of bone loss and the symptoms thereof .

24. A method for treating bone loss according to Claim 13 further comprising administering a therapeutically effective amount of PTH and lauramidopropyl betaine as separate dose units either simultaneously or sequentially within a therapeutically effective interval.

25. A method for treating bone loss comprising administering a pharmaceutical composition of PTH, betaine zwitterionic acid surfactant and a co-agent.

26. A method for treating bone loss comprising administering a pharmaceutical composition of PTH, a betaine zwitterionic acid surfactant and a co-agent as separate dose units either simultaneously or sequentially within a therapeutically effective interval.

27. A method for treating bone loss comprising administering a pharmaceutical composition of PTH, lauramidopropyl betaine zwitterionic acid surfactant and raloxifene™ as separate dose units either simultaneously or sequentially within a therapeutically effective interval .

28. The use of PTH, betaine zwitterionic acid surfactant and a co-agent in the manufacture of a medicament for the treatment and/or prophylaxis of bone loss .