COMPOSITIONS AND METHODS FOR ENHANCING SYSTEMIC DELIVERABILITY, TOLERABILITY, AND EFFICACY OF CATIONIC MACROCYCLIC PEPTIDES

Abstract

Compositions are provided for formulations of θ-defensin and/or a θ-defensin analog that are highly suitable for parenteral administration. Such formulations provide the θ-defensin and/or a θ-defensin analog in a slightly acidic buffer that includes propylene glycol. Surprisingly, Inventors have found that such formulation increase bioavailability of a θ-defensin and/or a θ-defensin analog so provided by at least a factor of 10 relative to conventional isotonic saline solutions, and that such formulations dramatically improved bioavailability in human subjects relative to animal models. Inventors have also found that such formulations advantageously exhibit low viscosity at high peptide concentrations, reducing injection volume permitting sterilization by simple filtration.

Claims

1-36. (canceled)

37. A method of treating an individual with a chronic inflammatory condition, comprising: providing a quantity of θ-defensin or θ-defensin analog as a first aqueous solution comprising the θ-defensin or θ-defensin analog and from 0.5% to 1.5% v/v propylene glycol, wherein the first aqueous solution has a pH of from 6.0 to 7.0; and administering the first aqueous solution by subcutaneous injection to an individual in need of treatment, wherein the first aqueous solution is formulated to provide an increase in pharmacologic potency or therapeutic effect of the θ-defensin or θ-defensin analog relative to a second aqueous solution comprising the quantity of θ-defensin or θ-defensin analog in a normal saline solution.

38. The method of claim 37, wherein the aqueous solution further comprises an acetate salt.

39. The method of claim 37, wherein pharmacodynamic effect of the quantity θ-defensin or θ-defensin analog is increased by at least 10-fold relative to the quantity of the θ-defensin or θ-defensin analog provided in a normal saline solution.

40. The method of claim 37, wherein pharmacodynamic effect of the quantity of the θ-defensin or θ-defensin analog is increased by at least 40-fold relative to the quantity of the θ-defensin or θ-defensin analog provided in a normal saline solution.

41. The method of claim 37, wherein the chronic inflammatory condition is selected from the group consisting of rheumatoid arthritis, inflammatory bowel disease, inflammation associated with cancer, diabetes, and a chronic disease characterized by dysregulated or unresolved chronic inflammation.

42. The method of claim 37, wherein the first aqueous solution comprises the θ-defensin or θ-defensin analog at concentrations of up to 15 mgmL.sup.−1.

43. The method of claim 37, wherein the θ-defensin analog is selected from the group consisting of a cyclic octadecapeptide, a cyclic heptadecapeptide, a cyclic hexadecapeptide, a cyclic pentadecapeptide, and a cyclic tetradecapeptide.

44. The method of claim 37, wherein the first aqueous solution comprises 1% v/v propylene glycol and 20 mM acetate, and has a pH of 6.5.

45. A method of sterilizing an aqueous θ-defensin preparation, comprising: providing a θ-defensin or θ-defensin analog in an aqueous buffer comprising the θ-defensin at a concentration of at least 1 mgmL.sup.−1 and propylene glycol at from 0.5% to 1.5% v/v; and passing the aqueous buffer through a filter having a pore size of 0.2 μm or less, wherein the concentration of propylene glycol is selected to provide the pharmaceutical composition with a viscosity of up to 105 centipoise.

46. The method of claim 45, wherein the aqueous buffer further comprises an acetate salt.

47. The method of claim 45, wherein the aqueous θ-defensin preparation has a pH of 6.0 to 7.0.

48. The method of claim 45, wherein the θ-defensin or θ-defensin analog is provided at 10 mgmL.sup.−1 or more.

49. A pharmaceutical composition for treatment of a chronic inflammatory condition, comprising: a θ-defensin or θ-defensin analog at a concentration of up to 20 mgmL.sup.−1 in an aqueous solution comprising 0.5% to 1.5% v/v propylene glycol, wherein concentration of propylene glycol is selected to provide the pharmaceutical composition with a viscosity of up to 105 centipoise, wherein the pharmaceutical composition has a pH of from 6.0 to 7.0 and is formulated for parenteral administration.

50. The pharmaceutical composition of claim 49, further comprising an acetate salt.

51. The pharmaceutical composition of claim 49, wherein the concentration of θ-defensin or θ-defensin analog is selected such that pharmacologic potency of the θ-defensin or θ-defensin analog is increased by at least 10-fold relative to a similar concentration of the θ-defensin or θ-defensin analog provided in a normal saline solution.

52. The pharmaceutical composition of claim 49, wherein the concentration of θ-defensin or θ-defensin analog is selected such that pharmacologic potency of the θ-defensin or θ-defensin analog is increased by at least 40-fold relative to a similar concentration of the θ-defensin or θ-defensin analog provided in a normal saline solution.

53. The pharmaceutical composition of claim 49, wherein the chronic inflammatory condition is selected from the group consisting of rheumatoid arthritis, inflammatory bowel disease, inflammation associated with cancer, diabetes, and a chronic disease characterized by dysregulated or unresolved chronic inflammation.

54. The pharmaceutical composition of claim 49, wherein parenteral administration is selected from the group consisting of subcutaneous injection, intramuscular injection, or intravenous injection.

55. The pharmaceutical composition of claim 49, wherein the θ-defensin analog is selected from the group consisting of cyclic octadecapeptide, a cyclic heptadecapeptide, a cyclic hexadecapeptide, a cyclic pentadecapeptide, and a cyclic tetradecapeptide.

56. The pharmaceutical composition of claim 49, wherein the pharmaceutical composition comprises 1% v/v propylene glycol and 20 mM acetate, and has a pH of 6.5.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1: FIG. 1 shows typical data demonstrating the effects of subcutaneous injection of RTD-1 in saline on an animal model of rheumatoid arthritis.

[0025] FIG. 2: FIG. 2 shows typical effects of subcutaneous injection of RTD-1 in saline containing 1% (v/v) propylene glycol on an animal model of rheumatoid arthritis.

[0026] FIG. 3: FIG. 3 shows typical effects of subcutaneous injection of RTD-1 in saline containing 1% (v/v) propylene glycol or 20 mM Na acetate containing 1% (v/v) propylene glycol on an animal model of rheumatoid arthritis.

[0027] FIGS. 4A and 4B: FIGS. 4A and 4B show graphs of plasma θ-defensin concentration (in ng/mL) vs time (in hours) for male and female rats, respectively, on day 1 of a study in which a θ-defensin formulation of the inventive concept is injected subcutaneously.

[0028] FIGS. 5A and 5B: FIGS. 5A and 5B show graphs of plasma θ-defensin concentration (in ng/mL) vs time (in hours) for male and female rats, respectively, on day 13 of a study in which a θ-defensin formulation of the inventive concept is injected subcutaneously.

[0029] FIGS. 6A and 6B: FIGS. 6A and 6B show graphs of plasma θ-defensin concentration (in ng/mL) vs time (in hours) for male and female rats, respectively, on day 41 of a study in which a θ-defensin formulation of the inventive concept is injected subcutaneously.

[0030] FIGS. 7A and 7B: FIG. 7A provides a graph depicting results of a C.sub.max vs. θ-defensin dose linearity study for male and female rats at day 1 of a study in which a θ-defensin formulation of the inventive concept is injected subcutaneously. FIG. 7B provides a graph depicting results of a C.sub.max vs. θ-defensin dose linearity study for male and female rats at day 41 of a study in which a θ-defensin formulation of the inventive concept is injected subcutaneously.

[0031] FIGS. 8A and 8B: FIG. 8A provides a graph depicting results of an AUC.sub.0-TLast vs. θ-defensin dose linearity study for male and female rats at day 1 of a study in which a θ-defensin formulation of the inventive concept is injected subcutaneously. FIG. 8B provides a graph depicting results of an AUC.sub.0-TLast vs. θ-defensin dose linearity study for male and female rats at day 41 of a study in which a θ-defensin formulation of the inventive concept is injected subcutaneously.

[0032] FIG. 9: FIG. 9 shows typical results for measurement of θ-defensin concentration (ng/mL) in plasma over time for different treatment groups in a human clinical trial in which a θ-defensin formulation of the inventive concept was injected subcutaneously.

[0033] FIGS. 10A and 10B: FIG. 10A shows a typical dependence of C.sub.max (ng/mL) on dose (μg/kg) of a θ-defensin formulation of the inventive concept that was injected subcutaneously in a human clinical trial. FIG. 10B shows a typical dependence of AUC.sub.0-TLast on dose (μg/kg) of a θ-defensin formulation of the inventive concept that was injected subcutaneously in a human clinical trial.

[0034] FIGS. 11A and 11B: FIG. 11A shows a typical dependence of C.sub.max (ng/mL) on total θ-defensin dose (mg) of a θ-defensin formulation of the inventive concept that was injected subcutaneously in a human clinical trial. FIG. 11B shows a typical dependence of AUC.sub.0-TLast on total θ-defensin dose (mg) of a θ-defensin formulation of the inventive concept that was injected subcutaneously in a human clinical trial.

DETAILED DESCRIPTION

[0035] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus, if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.

[0036] The inventive subject matter provides apparatus, systems and methods in which propylene glycol at from 0.5% to 1.5% in a slightly acidic (e.g. pH of 5 to 7) aqueous solution has, surprisingly, been found to increase the preclinical efficacy of a parenterally administered θ-defensin and/or θ-defensin analog by at least 20-fold relative to conventional neutral isotonic saline solutions. This permits convenient subcutaneous administration of θ-defensin in relatively small volumes (e.g. about 1 mL) in amounts that are effective in treating chronic inflammatory conditions such as rheumatoid arthritis, inflammatory bowel disease, or diabetes, or other conditions resulting from dysregulated inflammatory responses in humans. Addition of propylene glycol at these concentrations was also unexpectedly found to dramatically reduce the viscosity of concentrated (e.g. 10 mgmL.sup.−1 or higher) solutions of θ-defensin, permitting simple, convenient, and scalable sterilization by filtration. Further, the formulation of θ-defensin markedly reduced injection site reactions relative to those observed when isotonic saline was used as the formulation vehicle.

[0037] Suitable θ-defensins include native θ-defensins found in mammals expressing these peptides, and can also include one or more θ-defensins derived from untranslated genes present in some primate species (e.g. Homo sapiens). In some embodiments of the inventive concept one or more θ-defensin can correspond to those found in Macaca mulatta, for example RTD-1 (SEQ ID NO. 1), RTD-2 (SEQ ID NO. 2), RTD-3 (SEQ ID NO. 3), RTD-4 (SEQ ID NO. 4), RTD-5 (SEQ ID NO. 5), and/or RTD-6 (SEQ ID NO. 6). In other embodiments of the inventive concept one or more θ-defensin can correspond to those found in Papio anubis, for example BTD-1 (SEQ ID NO. 7), BTD-2 (SEQ ID NO. 8), BTD-3 (SEQ ID NO. 9), BTD-4 (SEQ ID NO. 10), BTD-5 (SEQ ID NO. 11), and/or BTD-6 (SEQ ID NO. 12), BTD-7 (SEQ ID NO. 13), BTD-8 (SEQ ID NO. 14), BTD-9 (SEQ ID NO. 15), and/or BTD-10 (SEQ ID NO. 16).

[0038] Within this application embodiments describing θ-defensins and uses thereof are inclusive of θ-defensin analogs. The term θ-defensin analog refers to a cyclic peptide having about 40%, 50%, 60%, 70%, 80%, 90% or greater sequence identity with a native θ-defensin peptide sequence. A θ-defensin analog can incorporate one, two, three, or more core features of a native θ-defensin. Exemplary core features include cyclic structure, the presence of one, two, three, or more disulfide bonds within the peptide (e.g. between pairs of cysteines of the analog), having a positive charge when in solution under physiological conditions, and the presence of beta pleated sheet secondary structure. Such θ-defensin analogs can include 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acids, and in some embodiments can incorporate non-naturally occurring amino acids. An analog of a θ-defensin can include one or more L-amino acid(s), one or more D-amino acid(s), and/or a mixture of L- and D-amino acids. In some embodiments non-peptide bonds can be utilized between adjacent amino acid residues of a θ-defensin analog. θ-defensin analogs can represent one or more deletion or substitution of amino acids of a native θ-defensin sequence. Such substitutions can be conservative (e.g. where the substituted amino acid(s) retain(s) charge, hydrophobicity, hydrophilicity, and/or steric properties of the native amino acid). In some embodiments θ-defensin analogs can include grafting or conjugation of non-peptide moieties, for example polyethylene glycol and/or other hydrophilic polymers, cell-receptor targeting moieties, and/or moieties that aid in processing/purification. Examples of suitable theta defensin analogs that are based on RTD-1 (SEQ ID NO. 1) are provided as SEQ ID NO. 17 (RTD-1-27), SEQ ID NO. 18 (RTD-1-28), and SEQ ID NO. 19 (RTD-1-29).

[0039] Compositions and methods for enhancing the pharmacologic effect of a θ-defensin and/or a θ-defensin analog utilized in the treatment of chronic inflammatory conditions (e.g. rheumatoid arthritis and/or diabetes) are provided herein. Such compositions incorporate from about 0.5% to about 1.5% propylene glycol in a mildly acidic aqueous solution (e.g. about pH 5 to 7), and are suitable for subcutaneous injection of θ-defensin at a concentration of up to 50 mgmL.sup.−1. Such formulations have, surprisingly, been found to dramatically (e.g. greater than at least 10-fold) increase in pharmacologic effect or potency of the administered θ-defensin relative to θ-defensin provided at the same or similar concentrations in conventional saline solutions and/or solutions that do not include propylene glycol. This is shown in FIGS. 1, 2, and 3 below. It has also been found that such formulations have greatly reduced viscosity relative to θ-defensin provided in conventional normal saline solutions (e.g. phosphate buffered saline, pH 7 to 7.5) at similar concentrations, which permits sterilization by filtration. Further, compositions claimed markedly reduce injection site reactions compared to isotonic saline vehicle.

[0040] FIGS. 1, 2, and 3 show the results of treatment of an animal model of rheumatoid arthritis by subcutaneous injection of the θ-defensin RTD-1, where the θ-defensin is provided in either a conventional normal saline vehicle or in normal saline containing a low concentration (1% v/v) of propylene glycol. Rats with established pristane induced arthritis were treated with daily subcutaneous injections of RTD-1 formulated in normal saline at the indicated doses (FIG. 1) or in a saline solution containing 1% propylene glycol (FIG. 2). The lowest effective RTD-1 dose in the normal saline diluent was 1 mg/kg, with maximal apparent effect seen at 3 mg/kg (see FIG. 1). The inclusion of 1% propylene glycol in the vehicle markedly lowered the effective RTD-1 dose (to 0.08 mg/kg, see FIG. 2) in that 0.08 mg/kg (the lowest dose tested) produced a maximal anti-arthritic effect, equivalent to 3 mg/kg doses of RTD-1 in saline (i.e. the maximum observed effect). Equivalent improvement in pharmacologic effect was obtained when RTD-1 was formulated in a buffered (20 mM sodium acetate) aqueous diluent containing 1% propylene glycol (FIG. 3). The Applicant believes that similar effects can be achieved at lower doses of the θ-defensin.

[0041] Surprisingly, it has also been found that such formulations have greatly reduced viscosity relative to θ-defensin provided in conventional normal saline solutions (e.g. isotonic phosphate buffered saline, pH 7 to 7.5) at similar concentrations, which permits sterilization by filtration.

[0042] Inventors have previously noted that θ-defensins and θ-defensin analogs (for example, cyclic octadecapeptides and/or cyclic tetradecapeptides) have significant effects in reducing inflammation in animal models of chronic inflammatory disease, such as pristane-treated rats. θ-defensins are readily soluble in aqueous solutions. In initial studies the θ-defensin was prepared in conventional normal saline solutions and was readily administered by subcutaneous injection with no apparent systemic ill effects in mice, though at high θ-defensin concentrations fat necrosis in the subcutaneous tissue was observed. Similarly, in rat and canine animal models, low concentrations of θ-defensins were well tolerated systemically and locally. However, at higher concentrations, injections of θ-defensin in normal saline resulted in localized inflammation and swelling at the injection site in a dose-dependent manner. Swelling and inflammation were found to persist for weeks. Relatively high concentrations of θ-defensin (e.g. about 10 to 50 mgmL.sup.−1), however, are likely to be necessary to provide the necessary θ-defensin dosage while maintaining volumes appropriate for subcutaneous injection in human therapy.

[0043] Microscopic studies of injection sites where θ-defensin had been administered in conventional saline solution showed localized inflammation and necrotic changes at injection sites of canine and porcine test subjects. Without wishing to be bound by theory, Inventors believe that θ-defensin may be interacting with components of the extracellular matrix found within the skin of some mammals at subcutaneous injection depths, causing it to precipitate and/or form precipitating complexes within this tissue layer. Studies of the interaction of RTD-1 with whole blood, blood plasma collected following treatment with anticoagulant, and serum indicate that the θ-defensin interacts with fibrinogen to form insoluble complexes. Inventors believe that θ-defensin may, therefore, be interacting with fibrinogen and/or fibrinogen-like proteins associated with the extracellular matrix.

[0044] Inventors found that use of hypotonic saline, addition of poloxamers, and the use of other conventional excipients failed to reduce or eliminate this θ-defensin induced injection site inflammation and tissue injury. Surprisingly, low concentrations of propylene glycol were effective in providing solubility for high concentrations (e.g. 10 mgmL.sup.−1 or greater) of θ-defensin and also in reducing or preventing swelling and/or inflammation on subcutaneous injection of high concentrations of θ-defensin into susceptible species. Propylene glycol is freely miscible with water, available as a sterile liquid of pharmaceutical grade, and is generally recognized as safe. The effective range of propylene glycol concentrations for reduction and/or elimination of adverse reactions on injection of high concentrations of θ-defensin was found to be relatively narrow. In some embodiments the effective concentration of propylene glycol in water was found to be from about 0.4% to about 1.6% (v/v). In other embodiments the effective concentration of propylene glycol in water was found to be about 0.5% to about 1.5% (v/v). In a preferred embodiment the effective concentration of propylene glycol in water is about 1% (v/v).

[0045] Further studies showed that, surprisingly, results were further improved by using a propylene glycol/water solvent with mildly acidic pH (e.g. from about pH 5 to 7). In a preferred embodiment the pH of the propylene glycol/water solvent is about 6.0, and has a final osmolality of about 180 to 230 mOsmoles. The pH of the propylene glycol/water solvent can be maintained using a buffer species at low molarity (e.g. less than about 50 mM). Suitable buffer concentrations can, for example, range from 1 mM to 50 mM, 5 mM to 35 mM, 10 mM to 30 mM, or about 20 mM. Suitable buffer species can be salts of organic acids, such as acetate, citrate, malate, tartrate, HEPES, MES, etc. In other embodiments the buffer species can be a zwitterionic species having a suitable pKa. In a preferred embodiment the buffer species is an acetate salt (e.g. sodium acetate) at a concentration of about 20 mM, and provides a pH of about 6.0. Surprisingly, Inventors have found that the use of saline (e.g. NaCl) in even small (e.g. less than 50 mM) amounts results in adverse reactions to subcutaneous injection of θ-defensin. Preferred embodiments of the aqueous solvent system used for subcutaneous administration can exclude NaCl and similar salts.

[0046] Surprisingly, Inventors have found that θ-defensins are freely soluble in such acidic propylene glycol/water solvent systems, and that only transient dermal reactions are found following subcutaneous injections of RTD-1 in rats and dogs at concentrations of up to 12.5 mgmL.sup.−1, and with only minor adverse effects at 50 mgmL.sup.−1. This supports the estimated 10 mgmL.sup.−1 concentration of θ-defensin expected as necessary for providing dosing adequate for human treatment.

[0047] As noted above, Inventors unexpectedly found that use of a propylene glycol/water solvent system, in addition to eliminating the inflammation associated with saline-based formulations, dramatically increases the pharmacologic potency of a θ-defensin administered subcutaneously relative to conventional saline formulations. The increase in preclinical efficacy in established pristane-induced arthritis in mildly acidic propylene glycol/water relative to similar amounts provided in conventional saline solutions can be at least 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, or more. For example, subcutaneous injection of RTD-1 provided at 0.08 mg/kg in 1% propylene glycol+20 mM acetate, pH 6, was found to have an effect equivalent to the administration of RTD-1 at 3 mg/kg in isotonic saline in the relief of symptoms of rheumatoid arthritis. This suggests an increase in pharmacologic potency of at least 37-fold, however since the relief of symptoms provided was at the upper limit of the benefit afforded by θ-defensins administered in isotonic saline it is probable that the actual increase in pharmacodynamic effect is greater than 37-fold (e.g. greater than 40-fold, 50-fold, 70-fold, 100-fold, or higher). This advantageously reduces the amount of θ-defensin necessary to provide adequate treatment.

[0048] Sterilization of conventional solutions utilized for injection is commonly performed by filtration, for example using a filter having a pore size of 0.2 μm or less. This process is, however, problematic for protein drug solutions as such solutions are typically too viscous for efficient sterile filtration at the high protein concentrations that are desirable to provide small injection volumes. As used herein, such a viscous solution refers to a solution or dispersion in which the internal resistance to flow is so high that filtration is difficult or impossible (e.g. requiring pressures exceeding the burst pressure of the filtration membrane). The viscosity of such a viscous solution can be as high as 105 centipoise (cp) or higher. In one embodiment the viscosity of such a viscous solution is at least about 90 to about 95 cp. In another embodiment the viscosity of such a viscous solution is at least about 40 cp. In still another embodiment the viscosity of such a viscous solution is at least above the viscosity of water (i.e. above about 1.0 cP).

[0049] During the course of investigation Inventors found that, while θ-defensins are highly soluble, aqueous solutions can become highly viscous as protein concentration increases. In practice conventional saline solutions of 2% w/v θ-defensin or higher were found to be too viscous to permit sterilization by filtration at a reasonable and/or manufacturable scale. Surprisingly, similar solutions prepared in propylene glycol/water solvents have low viscosity at high (e.g. 2% w/v, 5% w/v, 10% w/v, or greater) θ-defensin concentrations. This permits simple and scalable sterilization of such preparations using conventional filtration through media having a pore size of about 0.2 μm or less.

[0050] Embodiments of the inventive concepts include methods of treating conditions associated with chronic inflammation by subcutaneous injection of a drug composition that includes one or more θ-defensin(s) in an acidic aqueous solution containing propylene glycol at from 0.5% to 1.5%. Suitable conditions include rheumatoid arthritis, inflammatory bowel disease, diabetes, and other conditions resulting from dysregulation inflammatory responses. The drug composition can include a single species of θ-defensin or two or more species of θ-defensins. The drug composition can be administered subcutaneously in a volume ranging from about 0.1 mL to about 2.5 mL, about 0.25 mL to about 2 mL, 0.5 mL to about 1.5 mL, or about 1 mL. The concentration of θ-defensin or total concentration of species of θ-defensins in the drug composition can range from about 1 mgmL.sup.−1 to about 50 mgmL.sup.−1, and is preferably about 12.5 mgmL.sup.−1 or less. Concentration of θ-defensin and/or injection volume can be adjusted to provide a dose of from about 0.001 mg/kg to about 3 mg/kg, about 0.01 to about 1 mg/kg, or about 0.08 mg/kg.

[0051] In some embodiments the drug composition can include, in addition to one or more θ-defensin(s) and/or θ-defensin analog(s) and propylene glycol, additional therapeutic compounds. For example, the drug composition can include one or more steroids having an anti-inflammatory effect, non-steroidal anti-inflammatory drug(s), antibody(ies) or antibody fragment(s) directed to a pro-inflammatory cytokine, and/or one or analgesic compound(s).

[0052] Such preparations of θ-defensin can be administered using any suitable schedule. Subcutaneous injections can be provided at a frequency of once a week, twice a week, three times a week, alternating days, daily, every 12 hours, every 8 hours, or every 6 hours, as necessary to establish or maintain a desired therapeutic effect. Length of treatment can range from about 1 week, about 2 weeks, about 4 weeks, about 8 weeks, about 12 weeks, about 16 weeks, about 20 weeks, about 6 months, about 12 months, about 18 months, about 24 months, or greater than 24 months, as necessary to establish or maintain a desired therapeutic effect. In some embodiments dosing with θ-defensin can be higher and/or more frequent during initial stages of treatment in order to establish remission or partial remission of symptoms, then reduced in terms of either or both of θ-defensin dose and/or frequency of administration in order to maintain the remission of symptoms.

EXAMPLES

[0053] Pharmacokinetic Studies in Rats

[0054] The θ-defensin RTD-1 was formulated in a mildly acidic buffer containing propylene glycol as described above, and administered subcutaneously to male and female Sprague-Dawley rats followed by determination of plasma levels of the θ-defensin. Rats received doses ranging from 1 to 4 mg/kg. Dose volume was held constant at 0.32 mL/kg; accordingly, concentration of the θ-defensin ranged from 3.125 to 12.5 mg/mL. Doses were administered three times per week for six weeks. Exemplary results are shown in Table 1 (which provides results for male rats) and Table 2 (which provides results for female rats). Table 1 and the following tables use the following acronyms: [0055] AUC.sub.0-Last Area under the plasma concentration versus time curve from time 0 (pre-dose) to the last measurable concentration time point [0056] C.sub.Last Last plasma concentration measured above the limit of quantitation [0057] C.sub.max Maximum plasma concentration [0058] MRT.sub.Last Mean residence time up to the last time point when plasma concentrations of analyte were measured [0059] T.sub.Last Time at which the last plasma concentration above the limit of quantification was measured [0060] T.sub.max Time of maximum concentration.

TABLE-US-00001 TABLE 1 Group C.sub.max T.sub.max AUC.sub.0-TLast T.sub.Last C.sub.Last MRT.sub.Last and Dose (ng/mL).sup.1 (hr).sup.1 (ng*hr/mL) (hr) (ng/mL) (hr) 2) RTD-1 Day 1 Low Dose 14.951 12 159 12 14.951 6.4 (1 mg/kg/ Day 13 dose) 74.644 4.0 951 24 13.369 10.0 Day 41 164.846 12 2158 24 17.766 9.8 3) RTD-1 Day 1 Mid Dose 26.635 0.5 319 24 13.747 11.8 (2 mg/kg/ Day 13 dose) 29.429 0.5 512 24 23.851 12.6 Day 41 208.296 12 3462 24 80.526 10.8 4) RTD-1 Day 1 High Dose 28.403 0.5 451 24 22.182 12.7 (4 mg/kg/ Day 13 dose) 54.720 0.5 776 24 35.863 12.6 Day 41 435.68 24 8103 24 435.680 13.1 .sup.1The apparent C.sub.max and T.sub.max values were determined for blood sampling times between pre-dose and 24 hours post dosing. There was no blood sampling between 12 and 24 hours. On Day 41, one highest mean plasma concentration in the High Dose group was found at the 24-hour blood sampling time point, indicating that it was possible that T.sub.max was achieved between 12 and 24 hours.

TABLE-US-00002 TABLE 2 Group C.sub.max T.sub.max AUC.sub.0-TLast T.sub.Last C.sub.Last MRT.sub.Last and Dose (ng/mL).sup.1 (hr).sup.1 (ng*hr/mL) (hr) (ng/mL) (hr) 2) RTD-1 Day 1 Low Dose 18.901 0.5 357 24 14.579 12.2 (1 mg/kg/ Day 13 dose) 97.675 24 1215 24 97.675 15.4 Day 41 176.959 0.5 2133 24 144.416 13.8 3) RTD-1 Day 1 Mid Dose 20.997 12 400 24 13.849 12.0 (2 mg/kg/ Day 13 dose) 37.786 24 636 24 37.786 14.4 Day 41 296.145 12 4675 24 127.434 11.5 4) RTD-1 Day 1 High Dose 37.129 12 743 24 36.851 13.6 (4 mg/kg/ Day 13 dose) 177.471 12 2364 24 51.198 11.5 Day 41 305.575 12 6138 24 263.958 12.6 .sup.1The apparent C.sub.max and T.sub.max values were determined for blood sampling times between pre-dose and 24 hours post dosing. There was no blood sampling between 12 and 24 hours. On Day 41, one highest mean plasma concentration in the High Dose group was found at the 24-hour blood sampling time point, indicating that it was possible that T.sub.max was achieved between 12 and 24 hours.

[0061] Tables 3, 4, and 5 show typical results from kinetic studies of plasma concentration of the θ-defensin (in ng/mL) in male rats receiving 1 mg/kg, 2 mg/kg, and 4 mg/kg, respectively. Tables 6, 7, and 8 show typical results from similar studies performed in female rats. FIGS. 4A and 4B show graphs of plasma θ-defensin concentration (in ng/mL) vs time (in hours) for male and female rats, respectively, on day 1 of such a study. FIGS. 5A and 5B show graphs of plasma θ-defensin concentration (in ng/mL) vs time (in hours) for male and female rats, respectively, on day 13 of such a study. FIGS. 6A and 6B show graphs of plasma θ-defensin concentration (in ng/mL) vs time (in hours) for male and female rats, respectively, on day 41 of such a study.

TABLE-US-00003 TABLE 3 Sampling Time Time Post Dosing Relative to Pre-dose Group Pre-dose 30 min 1 hr 2 hr 4 hr 8 hr 12 hr 24 hr 2-TK RTD-1 Day 1 Low Dose Mean BLOQ 13.302 13.614 BLOQ 12.464 BLOQ 14.951 BLOQ (1 mg/kg/dose) SD N/A 2.630 3.329 N/A N/A N/A N/A N/A % CV N/A 20 24 N/A N/A N/A N/A N/A N* 0 3 2 0 1 0 1 0 Day 13 Mean 18.660 21.697 52.084 13.177 74.644 14.283 64.415 13.369 SD N/A 6.701 69.303 5.121 88.833 2.196 87.142 2.164 % CV N/A 31 133 39 119 15 135 16 N 1 3 3 3 2 3 3 3 Day 41 Mean 74.847 26.314 148.135 21.294 152.933 24.763 164.846 17.766 SD 97.607 8.487 181.751 10.080 179.737 7.553 208.874 5.992 % CV 130 32 123 47 118 31 127 34 N 3 3 3 3 3 3 3 3 BLOQ—Below the limit of quantitation, <10 ng/mL; N represents the number of rats reporting with RTD-1 concentrations above the limit of quantitation. Values are presented as the mean ± SD of N = 3 and the average ± the range of N = 2. N/A—not applicable.

TABLE-US-00004 TABLE 4 Sampling Time Time Post Dosing Relative to Pre-dose Group Pre-dose 30 min 1 hr 2 hr 4 hr 8 hr 12 hr 24 hr 3-TK RTD-1 Day 1 Mid Dose Mean BLOQ 26.635 22.732 13.967 11.224 14.136 11.934 13.747 (2 mg/kg/dose) SD N/A 1.214 15.855 0.404 1.575 0.892 2.022 2.301 % CV N/A 5 70 3 14 6 17 17 N 0 3 3 2 3 3 2 3 Day 13 Mean BLOQ 29.429 19.115 19.560 16.807 23.740 20.622 23.851 SD N/A 12.833 0.819 3.122 1.755 5.215 1.842 2.561 % CV N/A 44 4 16 10 22 9 11 N 0 3 3 3 3 3 3 3 Day 41 Mean 135.977 92.475 190.915 75.016 200.315 93.949 208.296 80.526 SD 114.179 59.333 143.297 45.629 157.574 44.884 179.603 44.818 % CV 84 64 75 61 79 48 86 56 N 3 3 3 3 3 3 3 3 BLOQ—Below the limit of quantitation, <10 ng/mL; N represents the number of rats reporting with RTD-1 concentrations above the limit of quantitation. Values are presented as the mean ± SD of N = 3 and the average ± the range of N = 2. For N = 3, bold values are significantly different from Day 1; pair-wise comparison, p < 0.05 Student's t-test. N/A—not applicable.

TABLE-US-00005 TABLE 5 Sampling Time Time Post Dosing Relative to Pre-dose Group Pre-dose 30 min 1 hr 2 hr 4 hr 8 hr 12 hr 24 hr 4-TK RTD-1 Day 1 High Dose Mean BLOQ 28.403 17.401 14.295 16.338 22.693 16.141 22.182 (4 mg/kg/dose) SD N/A 10.138 3.699 4.245 3.656 5.658 3.924 4.199 % CV N/A 36 21 30 22 25 24 19 N 0 3 3 3 2 3 3 3 Day 13 Mean 10.875 54.720 25.465 28.070 22.843 38.772 30.731 35.863 SD 0.745 12.698 6.083 1.916 2.595 10.251 10.583 12.180 % CV 7 23 24 7 11 26 34 34 N 2 3 3 3 3 3 3 3 Day 41 Mean 231.831 386.809 289.317 334.089 276.001 356.169 283.309 435.680 SD 140.748 360.928 190.507 367.103 161.971 363.267 154.004 533.685 % CV 61 93 66 110 59 102 54 122 N 3 3 3 3 3 3 3 3 BLOQ—Below the limit of quantitation, <10 ng/mL; N represents the number of rats reporting with RTD-1 concentrations above the limit of quantitation. Values are presented as the mean ± SD of N = 3 and the average ± the range of N = 2. For N = 3, bold values are significantly different from Day 1; pair-wise comparison, p < 0.05 Student's t-test. N/A—not applicable.

TABLE-US-00006 TABLE 6 Sampling Time Time Post Dosing Relative to Pre-dose Group Pre-dose 30 min 1 hr 2 hr 4 hr 8 hr 12 hr 24 hr 2-TK RTD-1 Day 1 Low Dose Mean BLOQ 18.901 15.549 12.780 12.731 14.212 16.814 14.579 (1 mg/kg/dose) SD N/A 7.198 2.879 N/A 1.960 4.152 2.611 6.260 % CV N/A 38 19 N/A 15 29 16 43 N 0 3 3 1 3 2 3 3 Day 13 Mean BLOQ 43.692 11.859 65.055 12.849 78.536 18.469 97.675 SD N/A 43.430 0.935 76.310 1.092 114.621 1.051 136.032 % CV N/A 99 8 117 9 146 6 139 N 0 3 3 2 2 3 3 3 Day 41 Mean 17.879 176.959 20.237 154.096 22.465 156.429 29.272 144.416 SD N/A 274.447 13.515 243.129 12.674 233.247 12.116 212.016 % CV N/A 155 67 158 56 149 41 147 N 1 3 3 3 3 3 3 3 BLOQ—Below the limit of quantitation, <10 ng/mL; N represents the number of rats reporting with RTD-1 concentrations above the limit of quantitation. Values are presented as the mean ± SD of N = 3 and the average ± the range of N = 2. N/A—not applicable.

TABLE-US-00007 TABLE 7 Sampling Time Time Post Dosing Relative to Pre-dose Group Pre-dose 30 min 1 hr 2 hr 4 hr 8 hr 12 hr 24 hr 3-TK RTD-1 Day 1 Mid Dose Mean BLOQ 16.871* 15.297 12.222 13.357 17.759* 20.997 13.849 (2 mg/kg/dose) SD N/A 5.493 3.621 0.856 0.350 1.003 6.238 2.506 % CV N/A 33 24 7 3 6 30 18 N 0 3 3 2 3 3 3 3 Day 13 Mean BLOQ 16.534 18.211 14.094 15.389 23.916 27.988 37.786* SD N/A 0.450 3.326 1.372 3.575 2.707 9.097 8.818 % CV N/A 3 18 10 23 11 33 23 N 0 3 3 3 3 3 3 3 Day 41 Mean 185.906 100.112 195.411 100.134 212.986 127.257 296.145 127.434 SD 264.656 138.637 253.419 137.944 280.436 166.411 411.433 161.438 % CV 142 138 130 138 132 131 139 127 N 3 3 3 3 3 3 3 3 BLOQ—Below the limit of quantitation, <10 ng/mL; N represents the number of rats reporting with RTD-1 concentrations above the limit of quantitation. Values are presented as the mean ± SD of N = 3 and the average ± the range of N = 2. For N = 3, bold values are significantly different from Day 1; pair-wise comparison, p < 0.05 Student's t-test. N/A—not applicable.

TABLE-US-00008 TABLE 8 Sampling Time Time Post Dosing Relative to Pre-dose Group Pre-dose 30 min 1 hr 2 hr 4 hr 8 hr 12 hr 24 hr 4-TK ORTD-1 Day 1 High Dose Mean BLOQ 19.827 17.019 14.163 23.085 27.873 37.129* 36.851 (4 mg/kg/dose) SD N/A 4.754 5.943 2.337 3.617 5.989 10.687 8.741 % CV N/A 24 35 16 16 21 29 24 N 0 3 3 3 3 3 3 3 Day 13 Mean 56.688 23.650* 109.634 19.566* 106.184 45.219 177.471 51.198 SD 66.312 2.089 135.109 1.845 131.529 14.302 207.364 21.121 % CV 117 9 123 9 124 32 117 41 N 3 3 3 3 3 3 3 3 Day 41 Mean 171.510 234.782 225.608 194.810 210.728 214.045 305.575 263.958 SD 120.089 319.717 159.433 270.617 150.795 267.542 263.070 352.308 % CV 70 136 71 139 72 125 86 133 N 3 3 3 3 3 3 3 3 BLOQ—Below the limit of quantitation, <10 ng/mL; N represents the number of rats reporting with RTD-1 concentrations above the limit of quantitation. Values are presented as the mean ± SD of N = 3 and the average ± the range of N = 2. For N = 3, bold values are significantly different from Day 1; pair-wise comparison. p < 0.05 Student's t-test. N/A—not applicable.

[0062] Table 9 shows typical results for dose proportionality in regard to C.sub.max and AUC.sub.0-TLast for male and female rats.

TABLE-US-00009 TABLE 9 Dose Dose C.sub.max C.sub.max AUC.sub.0-TLast AUC.sub.0-TLast Ratio Mid to Ratio High to Ratio Mid to Ratio High to Ratio Mid to Ratio High to Dose Day Low Dose Low Dose Low Dose Low Dose Low Dose Low Dose RTD-1, Male Day 1 2 4 1.78 1.90 2.01 2.84 Day 13 0.39 0.73 0.54 0.82 Day 41 1.26 2.64 1.60 3.75 RTD-1, Female Day 1 2 4 1.11 1.96 1.12 2.08 Day 13 0.39 1.82 0.52 1.95 Day 41 1.67 1.73 2.19 2.88

[0063] FIG. 7A provides a graph depicting results of a C.sub.max vs. θ-defensin dose linearity study for male and female rats at day 1. FIG. 7B provides a graph depicting results of a C.sub.max vs. θ-defensin dose linearity study for male and female rats at day 41. FIG. 8A provides a graph depicting results of an AUC.sub.0-TLast vs. θ-defensin dose linearity study for male and female rats at day 1. FIG. 8B provides a graph depicting results of an AUC.sub.0-TLast vs. θ-defensin dose linearity study for male and female rats at day 41.

[0064] Human Clinical Trials

[0065] The θ-defensin RTD-1 was formulated in a mildly acidic buffer containing propylene glycol as described above, and administered subcutaneously to male and female subjects ranging in age from 23 to 73 followed by determination of plasma levels of the θ-defensin. Subjects received a θ-defensin of 20 μg/kg, 40 μg/kg, 80 μg/kg, 160 μg/kg, or 325 μg/kg by subcutaneous injection. It should be appreciated that these doses are, relatively, substantially smaller than those used in the animal studies described above. Subjects receiving 20 to 80 μg/kg received doses delivered at a single site. Subjects receiving larger amounts received their doses distributed between two sites. FIG. 9 shows typical results for measurement of θ-defensin concentration (ng/mL) in plasma over time for the different treatment groups. As shown, bioavailability on subcutaneous injection is dramatically increased in humans relative to rats. Similarly improved results for human subjects were noted relative to canine and porcine animal models of subcutaneous injection with RTD-1. The dependence of C.sub.max (ng/mL) on RTD-1 dose (μg/kg) is shown in FIG. 10A. Results of a similar study of dependence of AUC.sub.0-TLast on RTD-1 dose (μg/kg) is shown in FIG. 10B. The dependence of C.sub.max (ng/mL) on total RTD-1 administered (mg) is shown in FIG. 11A. Results of a similar study of dependence of AUC.sub.0-TLast on total RTD-1 administered (mg) is shown in FIG. 10B. Both AUC.sub.0-TLast and C.sub.max are approximately linear functions of the θ-defensin dose. Remarkably, far lower doses of the θdefensin are required to reach similar plasma concentrations in humans than were noted in test animals. Inventors anticipate that similar or improved results will be observed with analogs of θ-defensin.

[0066] It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the spirit of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the specification claims refer to at least one of something selected from the group consisting of A, B, C . . . and N, the text should be interpreted as requiring only one element from the group, not A plus N, or B plus N, etc. filtration.