Vaginal systemic drug delivery

Abstract

The present invention relates to a therapeutically active compound for the treatment of a medical condition, wherein the therapeutically active compound is administered in liquid formulation via the vagina by using an intravaginal ring. The present invention further relates to a therapeutically active compound selected from the group consisting of oxybutynin and other anti-muscarinic compounds, gonadotropin-releasing hormone (GnRH) and derivatives, both agonists and antagonists, nitroglycerin and other directly or indirectly acting cGMP enhancers, buprenorphine and other agonistic, antagonistic or partial (ant)agonistic opioids, nicotine and derivatives, lorazepam and other benzodiazepines, insulin and other blood glucose regulating compounds, FSH and other hormones for ovulation stimulation, pramipexol and other dopamine agonists, oxytocin and other hypothalamic peptides for the treatment of a medical condition, wherein the therapeutically active compound is administered in liquid formulation via the vagina by using an intravaginal ring.

Claims

1. A method of administering a therapeutically active compound for the treatment of a medical condition, the method comprising: administering the therapeutically active compound in liquid formulation via the vagina by using an intravaginal ring configured to provide a detectable level in blood or plasma in 1 hour or less; wherein the intravaginal ring comprises a first rigid member having a first and second end, a second rigid member having a third and fourth end, a first flexible member coupled between the first and third ends, and a flexible part coupled between the second and fourth ends; wherein at least one of the first flexible member and the flexible part is at least partially elastic, wherein the elasticity of the at least one of the first flexible member and the flexible part is such that: the ring can be squeezed to transform a shape of the device from an extended shape to a collapsed shape for allowing the ring to be inserted into a vagina of a user; the device is pre-biased to assume the extended shape when little to no external force is being applied thereto, said extended shape corresponding to a substantially oval or annular ring shape; the device assumes a shape substantially corresponding to the extended shape when the device is placed and released at or near the fornix posterior vaginae of a user; wherein the first rigid member and/or second rigid member comprises a reservoir holding the therapeutically active compound to be delivered, an opening, and a pump for pumping the therapeutically active compound out of said opening; and a diagnostic mechanism for performing an intravaginal diagnosis or measurement therefor.

2. The method as claimed in claim 1, wherein the therapeutically active compound is selected from the group consisting of oxybutynin and other anti-muscarinic compounds, gonadotropin-releasing hormone (GnRH) and derivatives, both agonists and antagonists, nitroglycerin and other directly or indirectly acting cGMP enhancers, buprenorphine and other agonistic, antagonistic or partial (ant)agonistic opioids, nicotine and derivatives, lorazepam and other benzodiazepines, insulin and other blood glucose regulating compounds, FSH and other hormones for ovulation stimulation, pramipexol and other dopamine agonists, oxytocin and other hypothalamic peptides.

3. The method as claimed in claim 1, wherein the compound is GnRH and the medical condition is infertility, and the dosage administered by the intravaginal ring is between 10 μg and 2 mg per 90-120 minutes.

4. The method as claimed in claim 1, wherein the compound is oxybutinin and the medical condition is Overactive Bladder (OAB) and the daily dosage administered by the intravaginal ring is between 0.1 and 30 mg over divided doses.

5. The method as claimed in claim 1, wherein the compound is nitroglycerin and the medical condition is of cardiac or gynecological origin and the dosage administered by the intravaginal ring is between 0.1 and 10 mg per dose, depending on symptoms.

6. The method as claimed in claim 1, wherein the compound is buprenorphine and the medical condition is moderate to severe pain or opioid dependence and the dosage administered by the intravaginal ring is between 50 μg and 2 mg per day over divided doses.

7. The method as claimed in claim 1, wherein the compound is nicotine and the medical condition is smoking cessation or mild cognitive impairment and the daily dosage administered by the intravaginal ring is between 1 and 30 mg over divided doses.

8. The method as claimed in claim 1, wherein the compound is lorazepam and the medical condition is insomnia and the dosage administered by the intravaginal ring is between 0.1 and 10 mg, one before bedtime and one during sleep.

9. The method as claimed in claim 1, wherein the compound is insulin and the medical condition is diabetes and the dosage administered by the intravaginal ring is based upon individual patient sensitivity.

10. The method as claimed in claim 1, wherein the compound is FSH and/or an GnRH antagonist and/or LH and/or HMG and the medical condition is infertility and the dosage administered by the intravaginal ring is based upon individual patient sensitivity.

11. The method as claimed in claim 1, wherein the compound is pramipexole, for the medical indication Parkinson's disease and restless legs, or hyperprolactinemie and the dosage administered by the intravaginal ring is based upon individual patient sensitivity and varies between 0,3 and 10 mg/day.

12. The method as claimed in claim 1, wherein the compound is administered in a pulsatile manner or on-demand.

13. The method as claimed in claim 1, wherein the compound is a glucocorticoid for circadian administration in case of oncology.

14. The method as claimed in claim 1, wherein the compound is an immunotherapeutic compound for treatment of an oncological condition.

15. The method as claimed in claim 1, wherein the intravaginal ring further comprises one or more sensors for measuring parameters that can be used in the diagnosis of a medical condition.

16. The method as claimed in claim 15, wherein the sensor is selected from biochemical sensor, temperature sensor, glucose sensor, electromyogram (EMG) or pressure contraction sensor, cardiovascular sensor.

17. The method as claimed in claim 1, wherein the intravaginal ring comprises one or more of the following features: a battery, a transmitter configured for wireless transmission of measurement data corresponding to measurements performed by the sensor and/or measurement data or diagnosis information outputted by the diagnostic sensor, a receiver for wirelessly receiving control commands for remote control of at least one of the pump, the sensor, and the diagnostic device, a transceiver unit combining the receiver and transmitter.

18. The method as claimed in claim 1, wherein the intravaginal ring is configured to administer the therapeutically active compound in dosages of 50 μl.

19. The method as claimed in claim 1, wherein the intravaginal ring comprises a reservoir sized to contain a restricted amount of the therapeutically active compound in liquid formulation that ranges 1 ml to 3 ml.

Description

EXAMPLE

Introduction

(1) The inventors contemplated that the uptake of drugs by the vaginal mucosa could be highly efficient when compared to the oral route. Therefore, this route has the potential of being a first choice application route especially for drugs with low bioavailability, high first-pass effects, or drugs that are ideally administered in a pulsatile manner, exert local effects, or are needed on-demand, or can replace invasive administrations, like subcutaneous, intramuscular or others in chronic diseases. The absence of a first-pass effect may lead to more stable and uniform plasma levels and consequently better efficacy as well as allow lower dosages and thus lead to fewer adverse drug reactions and/or complications.

(2) In order to provide proof of concept, the pharmacokinetic behavior of six different water-soluble test compounds (oxybutynin, Gonadotropin-releasing hormone (GnRH), nitroglycerin, buprenorphine, nicotine and lorazepam) following vaginal application was investigated in Beagle dogs. Beagle dogs are the preferred animal model based on the availability of pharmacokinetic data for other (non-vaginal) dosing routes, allowing for a direct comparison of the vaginal dosing route with more conventional dosing routes.

(3) To study the pharmacokinetics of vaginal dosing of the six compounds to be tested, six groups of two female Beagle dogs received a dual vaginal administration at t=0 and t=125 minutes of either Oxybutynin HCl, GnRH, Buprenorphine, Nitroglycerin, Nicotine or Lorazepam. Blood samples were collected at pre-dose, 5, 10, 20, 30, 60, 120, 135, 155, 185 and 245 minutes after the first dose. Plasma samples were sent to the bioanalysis department of ABL, Assen, for determination of test compound levels by LC-MS/MS. Additional samples obtained from animals receiving Nitroglycerin were also analysed by the same laboratory in order to identify the concentrations of two major metabolites (1,2-Glyceryl dinitrate (1,2-GDN) and 1,3-Glyceryl dinitrate (1,3-GDN) of Nitroglycerin.

Materials and Methods

(4) Twelve female Beagle dogs (Harlan Winkle, Germany) that were ca. 4 months of age and had a body weight of ca. 4-8 kg at dosing time were used in this study. Thirteen days prior to the experimental start date the animals were acclimatized to the laboratory conditions. Upon arrival, the dogs were housed in a quarantine room and checked for overt signs of ill health and anomalies. Animals were kept in rooms ventilated with 9-11 air changes per hour and were maintained at a temperature of 15-21° C. and a relative humidity of 45-75% other than during room cleaning. Lighting was artificial with a sequence of 12 hours light and 12 hours dark. All animals were housed in subgroups of 6 dogs in suitable dog cages. Each dog was uniquely identified with a number that was programmed in a transponder, which was subcutaneously implanted at allocation. Each cage was provided with a card showing the animal identification numbers, the group code and the study code. Dogs were assigned to their groups based on bodyweight.

(5) The dogs received one portion of a commercial dog diet twice daily (in the morning and afternoon). Drinking water was offered ad libitum at all times.

Experimental Design

(6) The study comprised the groups described in the Table 2 below.

(7) TABLE-US-00002 TABLE 2 Nominal dose level Group 1 Test Article (mg/animal) No. dogs 1 Oxybutynin HCl .sup. 2 × 2.5 mg 2 2 2 Gonadotropin-releasing 2 × 0.5 mg 2 hormone (GnRH) 3 Nitroglycerin 2 × 0.3 mg 2 4 Buprenorphine 2 × 0.3 mg 2 5 Nicotine 2 × 5.0 mg 2 6 Lorazepam 2 × 0.8 mg 2
Conscious dogs were dosed vaginally with 50 μL of the formulated test substance at t=0 and t=125 minutes.

Test Substance Formulation

(8) The formulations were prepared as follows.

(9) Group 1: 50 mg/mL Oxybutynin HCl, prepared in-house

(10) A solution of 50 mg/mL was prepared in demineralized water. To this end, 50.7 mg of Oxybutynin HCl was weighed in an Eppendorf tube and was subsequently dissolved by adding 1014 mg demineralized water.

(11) Group 2: 10 mg/mL GnRH, prepared in house

(12) GnRH and its solvent were obtained from a local pharmacy. A solution of 10 mg/mL was prepared in 0.9% NaCl. To this end, to a vial containing 3.2 mg of GnRH 323 mg of a supplied 0.9% NaCl solution was added, which was then transferred into an Eppendorf tube. analysis.

(13) Group 3: 6.4 mg/mL Nitroglycerin, ready to use

(14) This solution was obtained from a local pharmacy. 1.0 mL of the ready-to-use solution was transferred to an Eppendorf tube by 19 spray releases from the supplied solution.

(15) Group 4: 6 mg/mL Buprenorphine, prepared in-house

(16) A solution of 6 mg/mL was prepared in demineralized water. This solution was prepared by weighing 6.0 mg of Buprenorphine in an Eppendorf tube and adding 999.2 mg demineralized water.

(17) Group 5: 100 mg/mL Nicotine, ready to use.

(18) A solution containing 100 mg/mL in propylene glycol was obtained from a Webshop. 1005 mg of this solution was transferred into an Eppendorf tube.

(19) Group 6: 16 mg/mL Lorazepam, prepared in-house

(20) A solution of 16 mg/mL was prepared in propylene glycol. To this end, 16.9 mg Lorazepam was weighed in an Eppendorf tube and 1086 mg of propylene glycol was added.

Sample Collection and Dose Formulations

(21) Blood samples of ca. 2 mL were collected at pre-dose and ca. 5, 10, 20, 30, 60, 120, 135, 155, 185 and 245 minutes after the first dose in K2-EDTA vials. Plasma samples were prepared, each sample was divided in two aliquots (one sample was shipped for analysis and one sample was kept as a back-up sample). These samples were stored at below −18° C.

(22) Dose solutions were prepared (groups 1, 2, 4 and 6) within 2 hours before dosing. The solutions were kept on ice, protected from light until dosing. Shortly before dosing, the dose solutions were allowed to adjust to room temperature.

(23) The animals received a vaginal dose of 50 μL via a positive-displacement pipette (which is a technically adequate method for dosing during a pilot experiment) at t=0 and t=125 minutes.

(24) Blood samples of ca. 2 mL were collected in K2EDTA vials from the vena jugularis at pre-dose and ca. 5, 10, 20, 30, 60, 120, 135, 155, 185 and 245 minutes after the first dose. Blood samples were kept on ice until further processing to avoid breakdown of the test substances.

(25) Blood was centrifuged at 4° C. for 10 min. at 2000× g between 20 min and 45 min after collection in order to prepare plasma samples. After centrifugation, the plasma was aliquoted into two cryovials and subsequently stored at below −18° C. until shipment to ABL.

(26) Each animal was observed twice daily (morning and afternoon) by cage-side observations. Body weights were determined during acclimatization and one day prior to dosing.

(27) Plasma samples were sent on dry ice to the bioanalytical department of ABL, Assen for determination of drug levels by LC-MS/MS. The analysis was applied to 22 plasma samples per test substance. Calibration samples, quality control samples and blank plasma samples were included. The analytical range was aimed at 0.1-100 ng/mL.

(28) The time schedule for dose administration and collection of blood samples as well as the actual time of dosing and blood sampling is given in Table 3 below. Blood samples were obtained using the Vacutainer system: ca. 2 mL of whole blood was collected in K.sub.2-EDTA vials before further processing.

(29) Below are the summaries of the methods that were applied for all 6 compounds that were tested.

Oxybutynin HCl

(30) TABLE-US-00003 Analytical Range 0.100-100 ng/mL LC system Shimadzu Nexera UPLC MS/MS system Sciex API5500 Sample volume 50 μL

Method Description

(31) Oxybutynin was extracted from dog K.sub.2-EDTA plasma by a liquid-liquid extraction with TBME. After liquid-liquid extraction the extract was evaporated under a stream of nitrogen and reconstituted in injection solvent. After preparation all samples were injected into the chromatographic system. Chromatographic separation was performed on a Acquity BEH C8 column using gradient elution. An API 5500 tandem mass spectrometer equipped with a Turbo Ion Spray probe operating in the positive multiple reaction monitoring mode was used for quantification.

GnRH

(32) TABLE-US-00004 Analytical Range 0.100-100 ng/mL LC system Shimadzu Nexera UPLC MS/MS system Sciex API5500 Sample volume 50 μL

(33) TABLE-US-00005 TABLE 3 Dosing Blood sampling Dose Animal 0 min 5 min 10 min 20 min 30 min 60 min 120 min Group number Dose 1 Actual BL2 Actual BL3 Actual BL4 Actual BL5 Actual BL6 Actual BL7 Actual 1 5 0:00 0:00 0:05 0:07 0:10 0:10 0:20 0:22 0:30 0:37 1:00 0:59 2:00 2:01 1 3 0:10 0:10 0:15 0:15 0:20 0:21 0:30 0:30 0:40 0:40 1:10 1:11 2:10 2:13 2 1 0:20 0:20 0:25 0:25 0:30 0:30 0:40 0:40 0:50 0:50 1:20 1:20 2:20 2:20 2 7 0:30 0:30 0:35 0:40 0:40 0:42 0:50 0:50 1:00 1:00 1:30 1:35 2:30 2:35 3 9 0:40 0:40 0:45 0:45 0:50 0:50 1:00 1:01 1:10 1:10 1:40 1:40 2:40 2:41 3 11 0:50 0:50 0:55 0:55 1:00 1:00 1:10 1:11 1:20 1:20 1:50 1:50 2:50 2:51 Dosing Blood sampling Dose Animal 125 min 135 min 155 min 185 min 245 min Group number Dose 2 Actual BL8 Actual BL9 Actual BL10 Actual BL11 Actual 1 5 2:05 2:05 2:15 2:13 2:35 2:39 3:05 3:04 4:05 3:59 1 3 2:15 2:15 2:25 2:25 2:45 2:45 3:15 3:15 4:15 4:23 2 1 2:25 2:25 2:35 2:35 2:55 2:57 3:25 3:25 4:25 4:25 2 7 2:35 2:36 2:45 2:51 3:05 3:09 3:35 3:39 4:35 4:35 3 9 2:45 2:45 2:55 2:57 3:15 3:15 3:45 3:45 4:45 4:45 3 11 2:55 2:55 3:05 3:05 3:25 3:25 3:55 3:56 4:55 4:55 Dosing Blood sampling Dose Animal 0 min 5 min 10 min 20 min 30 min 60 min 120 min Group number Dose 1 Actual BL21 Actual BL3 Actual BL4 Actual BL5 Actual BL6 Actual BL7 Actual 4 13 0:00 0:00 0:05 0:05 0:10 0:10 0:20 0:20 0:30 0:30 1:00 1:00 2:00 2:00 4 15 0:10 0:10 0:15 0:15 0:20 0:20 0:30 0:31 0:40 0:40 1:10 1:10 2:10 2:13 5 17 0:20 0:20 0:25 0:25 0:30 0:30 0:40 0:40 0:50 0:50 1:20 1:20 2:20 2:20 5 19 0:30 0:30 0:35 0:36 0:40 0:40 0:50 0:52 1:00 1:00 1:30 1:30 2:30 2:30 6 21 0:40 0:40 0:45 0:45 0:50 0:50 1:00 1:01 1:10 1:10 1:40 1:42 2:40 2:41 6 23 0:50 0:50 0:55 0:55 1:00 1:00 1:10 1:10 1:20 1:20 1:50 1:50 2:50 2:50 Dosing Blood sampling Dose Animal 125 min 135 min 155 min 185 min 245 min Group number Dose 2 Actual BL8 Actual BL9 Actual BL10 Actual BL11 Actual 4 13 2:05 2:05 2:15 2:15 2:35 2:35 3:05 3:07 4:05 4:05 4 15 2:15 2:15 2:25 2:25 2:45 2:45 3:15 3:17 4:15 4:15 5 17 2:25 2:25 2:35 2:35 2:55 2:57 3:25 3:25 4:25 4:25 5 19 2:35 2:35 2:45 2:46 3:05 3:05 3:35 3:35 4:35 4:35 6 21 2:45 2:45 2:55 2:58 3:15 3:16 3:45 3:46 4:45 4:48 6 23 2:55 2:55 3:05 3:05 3:25 3:25 3:55 3:55 4:55 4:55

Method Description

(34) GnRH was extracted from dog K.sub.2-EDTA plasma by a solid phase extraction with Oasis HLB columns. After solid phase extraction the extract was evaporated under a stream of nitrogen and reconstituted in injection solvent. After preparation, all samples were injected into the chromatographic system. Chromatographic separation was performed on an Acquity BEH C8 3.0×100 mm, 1.7 μm column using gradient elution. An API 5500 tandem mass spectrometer equipped with a Turbo Ion Spray probe operating in the positive multiple reaction monitoring mode was used for quantification.

Nitroglycerin

(35) TABLE-US-00006 Analytical Range 0.100-100 ng/mL LC system Shimadzu Nexera UPLC MS/MS system Sciex API4000 Sample volume 100 μL

Method Description

(36) Nitroglycerin was extracted from dog K.sub.2-EDTA plasma by a liquid-liquid extraction (LLE) with a mixture of dichloromethane and Methyl tert-Butyl ether. After LLE the extract was evaporated under a stream of nitrogen and reconstituted in injection solvent. After preparation, all samples were injected into the chromatographic system. Chromatographic separation was performed on a Thermo hypersil gold C18 column using gradient elution. An API4000 tandem mass spectrometer equipped with a Turbo Ion Spray probe operating in the negative multiple reaction monitoring mode was used for quantification.

Buprenorphine

(37) TABLE-US-00007 Analytical Range 0.100-100 ng/mL LC system Shimadzu Nexera UPLC MS/MS system Sciex API4000 Sample volume 50 μL

Method Description

(38) Buprenorphine was extracted from dog K.sub.2-EDTA plasma by a liquid-liquid extraction (LLE) with Methyl tert-Butyl ether. After the LLE the extract was evaporated under a stream of nitrogen and reconstituted in injection solvent. After preparation, all samples were injected into the chromatographic system. Chromatographic separation was performed on a Thermo Hypersil Gold column using gradient elution. An API4000 tandem mass spectrometer equipped with a Turbo Ion Spray probe operating in the positive multiple reaction monitoring mode was used for quantification.

Nicotine

(39) TABLE-US-00008 Analytical Range 0.100-100 ng/mL LC system Shimadzu Nexera UPLC MS/MS system Sciex API4000 Sample volume 50 μL

Method Description

(40) Nicotine and internal standard Nicotine-D3 were extracted from dog K.sub.2-EDTA plasma by precipitation with methanol. After precipitation, the samples were diluted with water and injected into the chromatographic system. Chromatographic separation was performed on a Waters XBridge™ C18 column using gradient elution. An API 4000 tandem mass spectrometer equipped with a TIS probe operated in the multiple reaction monitoring (MRM) in positive mode was used for quantification.

Lorazepam

(41) TABLE-US-00009 Analytical Range 0.100-100 ng/mL LC system Shimadzu Acquity UPLC MS/MS system Sciex API4000 Sample volume 50 μL

Method Description

(42) Lorazepam was extracted from dog K.sub.2-EDTA plasma by a liquid-liquid extraction with Methyl tert-Butyl Ether. After liquid-liquid extraction, the extract was evaporated under a stream of nitrogen and reconstituted in injection solvent. After preparation, all samples were injected into the chromatographic system. Chromatographic separation was performed on an Acquity UPLC BEH C8 column using gradient elution. An API4000 tandem mass spectrometer equipped with a Turbo Ion Spray probe operating in the positive multiple reaction monitoring mode was used for quantification.

(43) RESULTS

(44) The measured study sample results for all six test compounds are presented in Table 4.

Oxybutynin HCl

(45) Oxybutynin HCl (FIG. 1) was absorbed quickly following vaginal administration (T.sub.max either 10 or 20 min). Oxybutynin HCl was almost completely eliminated from the body at t=120 min. The second dose of Oxybutynin HCl resulted in similar concentrations for both animals. The maximal concentration observed (C.sub.max) value for one of the animals was more than 3-times higher than for the other, indicating variation in the vaginal absorption of Oxybutynin HCl in Beagle dogs. This might be due to variation in the exact site of dose application, although this procedure was standardized as much as possible. The fact that the relative shape of the concentration vs. time profiles for both dogs was similar after the first as well as the second administration suggests that individual differences in vaginal geometry and mucosal properties may play an important role. It is evident from Table 5 (see below) that acute vaginal administration of an aqueous solution of Oxybutynin HCl in the dog resulted in rapid absorption of the drug as well as plasma levels comparable to those seen in another study on dogs employing either a silastic intra-vaginal ring as well as those observed after either oral or dermal administration in humans (WO2011/163358). Peak levels in the present study were observed substantially faster than those following oral or transdermal administration (Kennelly M J, Rev. Urol., 2010; 12(1): 12-19).

(46) TABLE-US-00010 TABLE 4 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Oxybutynin HCl GnRH Nitroglycerin Buprenorphine Nicotine Lorazepam DOG 3 DOG 5 DOG 1 DOG 7 DOG 9 DOG 11 DOG 13 DOG 15 DOG 17 DOG 19 DOG 21 DOG 23 Dose solution 7.68 7.71 4.73 122 16.6 (mg/mL) Theoretical dose 2.01 0.38 0.39 0.24 6.10 0.83 administered (mg) Plasma Plasma Plasma Plasma Plasma Plasma concentration concentration concentration concentration concentration concentration (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) Pre-dose <LLOQ <LLOQ <LLOQ <LLOQ <LLOQ <LLOQ <LLOQ <LLOQ <LLOQ <LLOQ <LLOQ <LLOQ 5 min 2.28 9.42 1.35 0.22 <LLOQ <LLOQ 1.46 0.34 214 32.3 1.55 3.93 10 min 4.40 11.6 1.12 0.11 <LLOQ <LLOQ 6.82 1.22 138 23.0 2.65 3.47 20 min 4.08 14.1 0.41 <LLOQ <LLOQ <LLOQ 6.10 1.43 68.1 12.4 2.23 4.25 30 min 3.36 12.3 0.20 <LLOQ <LLOQ <LLOQ 6.59 1.25 41.6 7.87 1.49 3.28 60 min 1.76 6.21 <LLOQ <LLOQ <LLOQ <LLOQ 6.14 0.95 10.1 2.78 0.38 0.99 120 min 0.76 2.24 <LLOQ <LLOQ <LLOQ <LLOQ 2.83 0.35 1.67 0.77 <LLOQ 0.59 135 min 5.69 13.8 0.71 <LLOQ <LLOQ <LLOQ 3.56 0.83 168 111 7.65 7.13 155 min 4.00 15.6 <LLOQ <LLOQ <LLOQ <LLOQ 4.28 0.88 55.6 38.1 4.96 6.16 185 min 2.33 9.06 <LLOQ <LLOQ <LLOQ <LLOQ 4.84 0.67 14.7 16.2 1.89 3.32 245 min 1.17 3.92 <LLOQ 0.19 <LLOQ <LLOQ 2.75 0.43 2.04 2.51 0.35 1.10

(47) TABLE-US-00011 TABLE 5 Comparison of Oxybutynin HCl after two intravaginal doses with other dosing routes C.sub.max T.sub.max Species Dose Route (ng/mL) (min) Reference Dog 2.5 (1st) Vaginal 4.4 10 Present study Dog 2.5 (2nd) Vaginal 5.7 10 Present study Dog 2.5 (1st) Vaginal 14.1 15 Present study Dog 2.5 (2nd) Vaginal 15.6 20 Present study Dog 2.5 mg/24 h Vaginal 14.0 90 WO2011163358 (ring) Dog 6.0 mg/24 h Vaginal 18.8 90 WO2011163358 (ring) Dog 10 mg/24 h Oral 17.9 180 WO2011163358 Human 5.0 Oral 10.0 50 Kennelly.sup.1 Human 5.0 Oral 2.1 60 Kennelly.sup.1 (ext. rel.) Human 5.0 Dermal 4.2 1500 Kennelly.sup.1 (patch) Human 100   Dermal 3.2 1500 Kennelly.sup.1 (gel) .sup.1Kennelly MJ, Rev. Urol., 2010; 12 (1): 12-19

Gonadotropin-Releasing Hormone (GnRH)

(48) Gonadotropin-releasing hormone (GnRH) (FIG. 2) showed a very high turn-over in both animals. GnRH was rapidly absorbed in both animals after the first dose (T.sub.max=<5 minutes post-dosing). The elimination of GnRH was very fast as well, with plasma levels falling below the lower-limit-of-quantification (LLOQ) within 60 minutes for both animals. GnRH plasma levels following the second dose administration were lower than those observed after the first dose.

(49) Intra-vaginal GnRH administration in the present study was fast and resulted in plasma levels comparable to those seen in humans after intranasal dosing, as can be observed in Table 6. It is not known which plasma concentrations of GnRH are required for efficacy in e.g. Kallmann syndrome. However, it is anticipated that plasma levels obtained by intra-vaginal dosing might be sufficient in view of the fact that endogenous plasma levels are two orders of magnitude lower (see Table 6).

(50) TABLE-US-00012 TABLE 6 Comparison of GnRH after two intravaginal doses with other dosing routes C.sub.max T.sub.max Species Dose (mg) Route (ng/mL) (min) Reference Dog 0.5 (1st) Vaginal 1.4 5 Present study Dog 0.5 (2nd) Vaginal 0.7 10 Present study Dog 0.5 (1st) Vaginal 0.2 5 Present study Dog 0.5 (2nd) Vaginal 0.2 10 Present study Human 0.8 Intranasal 0.35 Unknown Handelsman.sup.1 Human Endogenous Endogenous 0.002 Not Araki.sup.2 .sup.1Handelsman DJ, et al., Endocr Rev, 1986; 7 (1), 95-105 .sup.2Araki S, et al., Endocrinol. Japan, 1986; 33 (4), 457-468

Nitroglycerin

(51) No parent compound concentrations could be detected in plasma from the animals receiving Nitroglycerin (FIG. 3a), which is likely caused by the very short half-life reported for this drug (Lee F W, et al., J Pharmacol Exp Ther, 1990; 255(3): 1222-1229). However, analysis of two well-known metabolites of Nitroglycerin (1,2- and 1,3-Glyceryl dinitrate) showed rapid formation of both metabolites, with peak concentrations at 5 or 20 minutes after dosing the parent compound. The second dose of Nitroglycerin resulted in (slightly) increased plasma concentrations for both metabolites.

(52) Compared to studies using a significantly higher oral dose of Nitroglycerin, the T.sub.max values observed for both metabolites in the present study were considerably shorter, although not as fast as those observed following intravenous dosing (Tables 7 and 8). Whereas the maximal concentrations obtained in the present study were not as high as those using the oral formulation, one should note that the dose used in the oral study was considerably higher.

(53) TABLE-US-00013 TABLE 7 Comparison of 1,2-GDN after two intravaginal doses with other dosing routes C.sub.max T.sub.max Dose (mg) (ng/mL) (min) Species Nitroglycerin Route 1,2-GDN 1,2-GDN Reference Dog 0.3 (1st) Vaginal 1.24 5 Present study Dog 0.3 (2nd) Vaginal 1.54 10 Present study Dog 0.3 (1st) Vaginal 0.64 20 Present study Dog 0.3 (2nd) Vaginal 1.73 10 Present study Dog 0.25 mg/kg Oral 85.4 28 Lee.sup.1 Dog 0.025 mg/kg Intravenous 25.5 3 Lee.sup.1 Dog 0.25 mg/kg Intravenous 150 4 Lee.sup.1 .sup.1Lee FW, et al., J Pharmacol Exp Ther, 1990; 255 (3): 1222-1229

(54) TABLE-US-00014 TABLE 8 Comparison of 1,3-GDN after two intravaginal doses with other dosing routes C.sub.max Dose (mg) (ng/mL) T.sub.max (min) Species Nitroglycerin Route 1,3-GDN 1,3-GDN Reference Dog 0.3 (1st) Vaginal 0.30 5 Present study Dog 0.3 (2nd) Vaginal 0.50 10 Present study Dog 0.3 (1st) Vaginal 0.28 20 Present study Dog 0.3 (2nd) Vaginal 0.50 10 Present study Dog 0.25 mg/kg Oral 55.7 27 Lee.sup.1 Dog 0.025 mg/kg Intravenous 2.82 3 Lee.sup.1 Dog 0.25 mg/kg Intravenous 37.8 7 Lee.sup.1 .sup.1Lee FW, et al., J Pharmacol Exp Ther, 1990; 255 (3): 1222-1229

Buprenorphine

(55) Buprenorphine (FIG. 4) was rapidly absorbed in both animals (T.sub.max at t=10 and 20 minutes, respectively). The elimination of Buprenorphine was relatively slow (at 120 minutes almost 50% of the C.sub.max was still found in plasma). Interestingly, the second administration of Buprenorphine did not result in higher C.sub.max values and also the T.sub.max was delayed when compared to the first dose. The plasma concentrations of Buprenorphine were more than four times higher for one animal than for the other one.

(56) Peak levels of Buprenorphine (Table 9) given intra-vaginally in the present study are in the same order as those observed following (higher) doses of Buprenorphine via subcutaneous administration (Nunamaker E A, et al., J Am Assoc Lab Anim Sci, 2014; 53(5): 494-501). In comparison with data obtained from studies in humans, the plasma concentrations observed in the present study are reached considerably faster and are considerably higher than those observed after buccal, sublingual or transdermal dosing in man. Only intravenous dosing in man is comparable to intra-vaginal dosing in the dog with respect to T.sub.max observed (Kuhlman J J, et al., J.Anal. Toxicol, 1996; 20: 369-378).

(57) TABLE-US-00015 TABLE 9 Comparison of Buprenorphine after two intravaginal doses with other dosing routes C.sub.max T.sub.max Species Dose (mg) Route (ng/mL) (min) Reference Dog 0.3 (1st) Vaginal 6.8 10 Present study Dog 0.3 (2nd) Vaginal 4.8 20 Present study Dog 0.3 (1st) Vaginal 1.4 15 Present study Dog 0.3 (2nd) Vaginal 0.9 20 Present study Dog 0.2 mg/kg Subcutaneous 19.6 17 Nunamaker.sup.1 Human 0.3 Buccal 0.47 Not RxList.com reported Human 0.4 Sublingual 0.65 90-360 Bullingham.sup.2 Human 10.0  Dermal 0.2 Not RxList.com reported Human 1.2 Intravenous 38.0 5 Kuhlmann.sup.3 Human 4.0 Sublingual 3.3 45 Kuhlmann.sup.3 Human 4.0 Buccal 2.0 45 Kuhlmann.sup.3 .sup.1Nunamaker EA, et al., J Am Assoc Lab Anim Sci, 2014; 53 (5): 494-501 .sup.2Bullingham RE, et al., Br J Clin Pharmacol, 1982; 13 (5): 665-673 .sup.3Kuhlman JJ, et al., J.Anal. Toxicol, 1996; 20: 369-378

Nicotine

(58) Nicotine (FIG. 5) showed very fast absorption profiles with T.sub.max=<5 minutes for both animals, the C.sub.max for one of the animals was more than 4-times higher than the C.sub.max observed for the other animal, which was also confirmed by differences in clinical signs observed for both animals. After the second dose, absorption was very fast again. Following the second dose the C.sub.max values for both animals were similar. The elimination of nicotine was again very fast: the plasma concentrations returned to baseline levels within 120 minutes after the second dose.

(59) Intra-vaginally applied nicotine is absorbed very rapidly (T.sub.max at 5 and 10 min, the first sampling points) and efficiently. Peak levels are comparable to those obtained after smoking or gum chewing (see Table 10 for comparison) in humans. Very rapid absorption is a prerequisite for “smoking-like reinforcing properties” of nicotine, thus increasing its efficacy in smoking cessation.

(60) TABLE-US-00016 TABLE 10 Comparison of Nicotine after two intravaginal doses with other dosing routes C.sub.max T.sub.max Species Dose (mg) Route (ng/mL) (min) Reference Dog 2.5 (1st) Vaginal 214 5 Present study Dog 2.5 (2nd) Vaginal 168 10 Present study Dog 2.5 (1st) Vaginal 32 5 Present study Dog 2.5 (2nd) Vaginal 111 10 Present study Dog 75 Oral 14 120 Matsushima.sup.1 Dog 33 Oral 15 60 Matsushima.sup.1 Dog 31 Oral 26 60 Matsushima.sup.1 Dog 148 Oral 36 60 Matsushima.sup.1 Dog 64 Oral 27 120 Matsushima.sup.1 Dog 57 Oral 32 30 Matsushima.sup.1 Human 2.0 Intrapulmonary 49 2 Russell.sup.2 Human 4.0 Oral (gum) 40 30 Russell.sup.2 .sup.1 Matsushima D, et al., J Pharm Sci, 1995; 84 (3): 365-369; .sup.2 Russell MA, et al., Br Med J, 1976; 6017, 1043-1046

Lorazepam

(61) Lorazepam (FIG. 6) administration resulted in similar C.sub.max values for both animals after both dose administrations, although the second administration resulted in higher C.sub.max. The T.sub.max values after both administrations were 10 and 20 minutes for both animals. After both administrations, the plasma concentration of Lorazepam returned to baseline within 120 min after administration. Similar Lorazepam concentrations were observed for both animals.

(62) Intra-vaginal administration of lorazepam resulted in rapid peak levels that are only slightly below peak levels in humans after various routes of administration (Table 11). Except for IV, the other dosing routes in humans appear to have considerably higher T.sub.max values.

(63) TABLE-US-00017 TABLE 11 Comparison of Lorazepam after two intravaginal doses with other dosing routes Dose C.sub.max T.sub.max Species (mg) Route (ng/mL) (min) Reference Dog 0.8 (1st) Vaginal 4.0 5 Present study Dog 0.8 (2nd) Vaginal 7.0 10 Present study Dog 0.8 (1st) Vaginal 2.8 10 Present study Dog 0.8 (2nd) Vaginal 7.8 10 Present study Human 2.0 Oral 33.4 55 Blin1 Human 2.0 Intravenous 47.6 6 Wermeling2 Human 2.0 Intramuscular 22.6 180 Wermeling2 Human 2.0 Nasal 33.4 55 Wermeling2 1Blin O, et al., Clinical Neuropharmacology, 2001; 24, 2: 71-81 2Wermeling DPH, et al., J Clin Pharmacol, 2001; 41: 1225-1231

CONCLUSIONS

(64) In conclusion, absorption after intra-vaginal administration was confirmed for all test compounds, including GnRH, a native peptide with a molecular weight of 1212. For Nitroglycerin, no detectable concentrations of the parent compound could be observed. However, analysis of two well-known metabolites showed rapid and efficient absorption, implying fast Nitroglycerin absorption as well.

(65) Whereas most compounds (Oxybutynin HCl, GnRH, Buprenorphine and Nicotine) showed relatively large differences in observed C.sub.max values in the two animal, lorazepam administration showed similar concentration profiles for both animals tested. In general, the T.sub.max values varied only slightly between animals within groups, and for most of the test compounds the absorption was very fast, making the vaginal route an interesting alternative for more conventional dosing routes.

(66) All in all, the results of the present study indicate (very) fast absorption for all 6 compounds tested. It should be noted that the Cmax at the time of the first blood sampling of 5 minutes (and subsequent lower concentrations at later samplings) certainly missed the exact Tmax that must have been earlier than at the 5 minute point. Furthermore, the very fast physiological and systemic reaction of the dogs after administration of nitroglycerin, nicotine and buprenorphine gave the earliest clinical indications of fast systemic vaginal absorption of the compounds in the circulatory system.