Composition for use in the treatment and/or prevention of infertility

Abstract

Compositions for treatment and/or prevention of infertility include a pollen extract and/or a pistil extract primarily obtained from plants belonging to the Pinaceae and/or Poaceae family.

Claims

1. A method of treatment of infertility in men with oligospermia, cryptozoospermia, azoospermia, asthenospermia, oligoasthenospermia and/or teratospermia, the method comprising: providing an oral composition comprising a pollen extract and a pistil extract, said pollen and pistil being obtained from plants belonging to the Pinaceae and/or Poaceae family under aqueous extraction at a temperature between 30° C. and 45° C., wherein the pollen extract comprises protein pollen allergen Lol p4 from Dactylis glomerata L. and the pollen extract and the pistil extract comprise beta-1,3-glucanase from Zea mays L.; administering a daily dose to a man with oligospermia, cryptozoospermia, azoospermia, asthenospermia, oligoasthenospermia and/or teratospermia.

2. The method of claim 1, wherein the oral composition is in the form of a tablet, capsule, soft gel, semi-solid, solid, liquid, or powder.

3. The method of claim 1, wherein the composition is in capsule form and has a weight of 650 mg.

4. The method of claim 1, wherein the daily dose is administered for a period of at least three months.

5. The method of claim 1, wherein the infertility is not associated with inflammation of a microbacterial origin.

6. The method of claim 1, wherein said pollen and pistil are obtained from plants belonging to the species Secale cereale L., Zea mays L., Pinus sylvestris L., Dactylis glomerata L., or mixtures thereof.

7. The method of claim 6, wherein the composition comprises: an aqueous pollen extract from Secale cereale L.; an aqueous pollen extract from Zea mays L.; an aqueous pollen extract from Pinus sylvestris L.; an aqueous pollen extract from Dactylis glomerata L.; and an aqueous pistil extract from Zea mays L.

8. The method of claim 3, wherein the capsule comprises a pollen and pistil extract from Dactylis glomerata L., Secale cereale L., and Zea mays L. and a pollen extract from pollens comprising Zea mays L., Secale cereale L., and Pinus sylvestris L.

9. The method of claim 1, wherein the oral composition comprises at least one amino acid, a carotenoid, a trace element, a vitamin and/or a root extract, taken alone or as a mixture.

10. The method of claim 9, wherein: the amino acid is lysine, methionine, or carnitine; the carotenoid is lycopene; the trace element comprises zinc and/or selenium; the vitamin is selected from one or more of vitamin B6, vitamin B9, vitamin B12, vitamin C, vitamin D and vitamin E; and the root extract is a maca root extract (Lepidium meyenii).

11. The method of claim 10, wherein the trace element is zinc.

Description

EXAMPLE 1

Inventive Compositions

(1) i. Composition A:

(2) Dosage form: Tablet

(3) Tablet weight: approx. 380 mg

(4) Aqueous pollen extract: about 40% by weight based on the total weight of the composition

(5) TABLE-US-00001 TABLE 1 Ingredients of the extract expressed by weight based on the total weight of the final pollen extract: Ingredients Quantity % Secale cereale L. Pollen 70-80 Zea mays L. Pollen 15-40 Dactylis glomerata L. Pollen  5-10

(6) Other ingredients in the final product, for example, include: microcrystalline cellulose, silicon dioxide, magnesium stearate and/or coating agents. Talc and shellac were used as a coating agent. The recommended daily dose is two tablets per day, to be taken in the morning or evening.

(7) The pollen extract for the above tablet was obtained using a conventional process for the preparation of pollen and/or pistil extracts.

(8) ii. Composition B:

(9) Dosage form: Capsule

(10) Tablet weight: approx. 380 mg or 640 mg

(11) Aqueous pollen extract: about 40% by weight based on the total weight of the composition

(12) TABLE-US-00002 TABLE 2 Ingredients of the extract expressed by weight based on the total weight of the final pollen and pistil extract: Ingredients Quantity % Zea mays L. Pollen 15-80 Pinus sylvestris L. Pollen 0.05 to 25.00 Zea mays L. Pistil  1-15

(13) Other ingredients in the final product, for example, include: microcrystalline cellulose, silicon dioxide, magnesium stearate and/or coating agents. Talc and shellac were used as a coating agent. The recommended daily dose is one or two tablets per day, to be taken in the morning or evening.

(14) The pollen and pistil extract for the above tablet was obtained using a conventional process for the preparation of pollen and/or pistil extracts.

(15) iii. Composition C:

(16) Dosage form: Tablet

(17) Tablet weight: approx. 380 mg or 640 mg

(18) Aqueous pollen extract: about 40% by weight based on the total weight of the composition

(19) TABLE-US-00003 TABLE 3 Ingredients of the extract expressed by weight based on the total weight of the final pollen and pistil extract: Ingredients Quantity % Secale cereale L. Pollen 45-90  Zea mays L. Pollen 1-35 Pinus sylvestris L. Pollen 0.01 to 5.00 Dactylis glomerata L. Pollen 3-30 Zea mays L. Pistil 0.1-10.sup. 

(20) Other ingredients in the final product, for example, include: microcrystalline cellulose, silicon dioxide, magnesium stearate and/or coating agents. Talc and shellac were used as a coating agent. The recommended daily dose is two tablets per day, to be taken in the morning or evening.

(21) The pollen and pistil extract for the above tablet was obtained using a conventional process for the preparation of pollen and/or pistil extracts.

(22) iv. Composition D:

(23) Dosage form: Tablet

(24) Tablet weight: approx. 380 mg or 640 mg

(25) Aqueous pollen extract: about 40% by weight based on the total weight of the composition

(26) TABLE-US-00004 TABLE 4 Ingredients of the extract expressed by weight based on the total weight of the final pollen and pistil extract: Ingredients Quantity % Secale cereale L. Pollen 45-90  Zea mays L. Pollen 1-35 Pinus sylvestris L. Pollen 0.01 to 5.00 Dactylis glomerata L. Pollen 3-30 Zea mays L. Pistil 0.1-10.sup. 

(27) Other ingredients in the final product, for example, include: microcrystalline cellulose, silicon dioxide, magnesium stearate and/or coating agents. Talc and shellac were used as a coating agent. The recommended daily dose is two tablets per day, to be taken in the morning or evening.

(28) The pollen and pistil extract for the above tablet was obtained using the process, which is the object of the invention, detailed in Example 2 below.

(29) v. Composition E:

(30) Dosage form: Tablet

(31) Tablet weight: approx. 650 mg

(32) Aqueous pollen extract: about 40% by weight based on the total weight of the composition

(33) TABLE-US-00005 TABLE 5 Ingredients of the extract expressed by weight based on the total weight of the final pollen and pistil extract: Ingredients Quantity % Secale cereale L. Pollen 45-90  Zea mays L. Pollen 1-35 Pinus sylvestris L. Pollen 0.01 to 5.00 Dactylis glomerata L. Pollen 3-30 Zea mays L. Pistil 0.1-10.sup. 

(34) In addition, the final product contains approximately 2-10 mg of zinc and other ingredients such as microcrystalline cellulose, silicon dioxide, vitamin E, magnesium stearate and/or coating agents. Talc and shellac were used as a coating agent. The recommended daily dose is one tablet per day, to be taken in the morning or evening.

(35) The pollen and pistil extract for the above tablet was obtained using the process, which is the object of the invention, detailed in Example 2 below.

(36) The presence of reticuline oxidase, endochitinase A, beta-1,3-glucanase, exopolygalacturonase, and non-specific lipid transfer protein was confirmed with LC-MS/MS.

(37) The compositions according to the invention are used in the treatment and/or prevention of infertility, particularly in men.

(38) Preferably, the composition, which is the object of the invention, is intended for the treatment and/or prevention of infertility in men with oligospermia, cryptozoospermia, azoospermia, asthenospermia, oligoasthenospermia and/or teratospermia.

(39) More preferably, the composition, which is the object of the invention, is intended for the treatment of infertility in men presenting, in particular, with oligoasthenospermia.

(40) Even more preferably, the composition, which is the object of the invention, is intended for the treatment of infertility in men with asthenospermia and/or teratospermia.

EXAMPLE 2

Production of a Spray-Dried Extract According to an Aspect of the Present Invention

(41) The process for the preparation of the extract described here is an aqueous extraction, allowing a controlled selection of the water-soluble proteins of interest. The chronology of the operations and the control of the manufacturing parameters allow the preservation of specific proteins or peptides that are characteristic of the species used. This ensures that the species in the extract can be traced. The titre of the extract is advantageously standardised in amino acids at its last stage of manufacture, during nebulisation. By this control of the manufacturing parameters, the traceability, standardisation of the amino acid titre and intra-batch reproducibility, framed by an extract specification, are ensured. i. Preparation of pollen and pistil extracts according to the innovative process: The appropriate time for harvesting is: June-July for rye pollen (Secale cereale L.) and cocksfoot pollen (Dactylis glomerata L.); July-September for maize pollen and pistil (Zea mays L.); May-June for Scots pine pollen (Pinus sylvestris L.).

(42) Pine pollen is harvested from the wild, the other pistils and pollen come from agricultural crops.

(43) Pollens and pistils are dried. The process can be characterised by the following main steps: a) aqueous extraction (preferably water and/or surfactant) of pollen; b) aqueous extraction (preferably water and/or surfactant) of pollen and pistil; c) spray-drying of the extracts obtained in steps (a) and (b) above; and d) recovery of said plant pollen and pistil extracts obtained in (c).

(44) Surprisingly, the Applicant was able to identify that, by modifying the operating conditions of the process for preparing the extracts intended for developing the inventive compositions, it was possible to increase the quality of the extracts. ii. Details of the main steps of the preferred process according to the invention: a. Extraction steps:

(45) The Applicant was able to demonstrate that the temperature of the extractions should be between 30° C.-45° C.

(46) Extraction is preferably carried out under continuous agitation for 12 to 90 hours. b. Spray-drying step of the extract mixture:

(47) The different extracts, preferably previously evaporated, filtered and/or decanted, are mixed to obtain a dry-substance content between 30% and 50%.

(48) The starting temperature of the spray-drying stage is between 138° C. and 168° C.

EXAMPLE 3

Comparison of C and D Compositions from Two Different Processes

(49) Composition D, obtained by the method described above (example 2), is compared with composition C, obtained by the prior art method, for which the extraction temperature is strictly above 45° C.

(50) The Applicant noted that the change in extraction temperature and physical separation conditions affect the result of the identification tests.

(51) The protein Q5TIW3 (marker of Dactylis glomerata L. pollen) is not present in the batches of extracts obtained by the prior art method, whereas it is present in the batches of extracts obtained by the method described in Example 2 above.

(52) Species cannot be traced with the old process.

(53) The development of the process is a response to a requirement for traceability and reproducibility while integrating industrial economic constraints. The search for markers/tracers in the extracts manufactured according to the prior art and those manufactured with the inventive process shows that the prior art processes do not make it possible to ensure that the species used can be tracked.

(54) TABLE-US-00006 TABLE 6 Markers or tracers present in two compositions whose extracts are obtained by two different processes: Extract markers from the Extract markers from the process according to process according to the Species example 9 (T ° < 45° C.) prior art (T ° C. > 45°) Pollens from Q5TIW8 - pollen allergen Q5TIW8 - pollen allergen Secale cereale L. Sec 4 Sec 4 Pollens and pistils from B6T5D7 - Reticulase B6T5D7 - Reticulase Zea Mays L. oxidase oxidase E1AFV5 - Beta-13- E1AFV5 unidentified glucanase Pollens from Dactylis Q5TIW3 Unidentified glomerata L. pollen allergen LoI p4 Pollens from Pinus Lipid transfer protein, Lipid transfer protein, sylvestris L. positive immunological positive immunological response response

EXAMPLE 4

Effectiveness of Inventive Compositions

(55) i. Tested compositions:

(56) In this example, the effects of compositions whose pollen and pistil extracts are obtained by the inventive process (composition D above) have been compared with prior art compositions. ii. Observational study process:

(57) The aim of this study is to evaluate the impact of a six-month treatment with composition D or with other prior art compositions (substantially of the same dosage form, the same weight and the same concentration of pollen extract in the composition) including pollen extracts (EA10P5, EA5P2, etc.) on the main spermatic parameters (cf. see below) in a population of men in consultation for a fertility problem and who are more precisely presenting with oligospermia, asthenospermia or oligoasthenospermia.

(58) We are focusing on analysing the impact of such a treatment, particularly in terms of sperm count and/or sperm motility in the patients included in the study.

(59) Unless otherwise specified, at the start of treatment visit, the compositions are given to the patient for a six-month course of one dose per day. The recommended daily dose is two tablets per day, to be taken in the morning or evening. Intake starts the same day.

(60) During the final visit of the trial, a post-treatment spermogram is performed. Treatment adherence is also assessed.

(61) Surprisingly, it has been shown that three months of administration of composition D improves semen quality more than a prior art composition, especially with regard to general sperm motility.

(62) In fact, it has been observed that motility is improved by at least 15%, whereas it is only improved by about 10% with a composition including EA10P5 pollen.

(63) Similarly, it has been shown that composition D improves sperm vitality, whereas it is reduced by about 15% with a composition comprising EA10P5 pollen.

(64) Furthermore, the Applicant observed that composition D, which is the object of the invention, also improves certain disorders such as sexual dysfunction, decreased libido, erectile dysfunction, and premature or delayed ejaculation.

(65) The Applicant also compared the effects of composition D, which is the object of the invention, on semen parameters with another composition comprising palm pollen (Phoenix dactylifera). The Applicant was therefore able to show that composition D improves the number of spermatozoa per ml of semen by more than 60% after six months of treatment, whereas it is only improved by about 50% with an equivalent composition comprising palm pollen (Phoenix dactylifera).

(66) Finally, the Applicant compared the different parameters of a spermogram of 25 patients before and after treatment with composition D for a period of six months.

(67) The Applicant observed that the parameters of the spermogram were improved, including the following parameters: Volume pH Count Motility Morphology Vitality Leukocytes

(68) All of these studies therefore highlight an unexpected potentiated effect in a patient population with the administration of inventive composition D, which significantly increases the chances of finding fertile spermatozoa in the ejaculate by enabling the generation of the output of motile spermatozoa in hypofertile or even infertile men presenting with both oligospermia and asthenospermia, and therefore producing no or substantially no fertile spermatozoa.

(69) All patients evaluated in the study responding favourably to treatment with inventive composition D now produce a sufficient concentration of spermatozoa with adequate motility per ml of ejaculate to provide natural fertilisation, and thus avoid the situation whereby the patients must turn to sperm donation or adoption to have a child.

EXAMPLE 5

Spermogram Parameter Results Obtained after Treatment with Inventive Composition E on Infertile Patients

(70) During the first enrolment visit, patients undergo a spermogram before treatment. At the start of treatment, the product is given to them for a treatment of one daily intake for three months. The recommended daily dose is one tablet per day, to be taken in the morning or evening. Intake starts the same day. During the final visit of the trial, a post-treatment spermogram is performed. Treatment adherence is also assessed. The sampling conditions are strict. Semen collection is done by masturbation. Semen is collected after two to four days of sexual abstinence, after careful disinfection of the glans, and when the subject is not experiencing a period of fever.

(71) Comparative results (before and after treatment) are detailed below: i. Information about the patient(s) whose results are listed in Table 7 below: non-smoker grade 2/3 left varicocele (Varicocele is characterised by the dilation of a vein (varicose vein) in the spermatic cord, a fibrous cord located in the sacs above each testicle, which connects each one to the scrotum) no other treatment is taken no vitamin supplementation at the start of treatment

(72) TABLE-US-00007 TABLE 7 2010 WHO Before After PARAMETERS Standards treatment treatment volume >1.5 ml >1.5 ml >1.5 ml pH 7.2-8 7.2-8 7.2-8 Count 15 million/ml 10 million/ml no change Motility >40% total motility 26% total 35% total >32% progressive motility motility motility Morphology >4% according to >4% according >4% according Kruger to Kruger to Kruger Vitality >58% >58% >58% leukocytes <1 million/ml <1 million/ml <1 million/ml

(73) Conclusion on the results presented in Table 7:

(74) Before treatment, sperm concentration is low (10 million/ml) and there is a hypo-motility of 26%. The other parameters are normal. After treatment, the sperm concentration remains unchanged, but the motility is increased to 35%. No adverse effects are reported. ii. Information about the patient(s) whose results are listed in Table 8 below: smoker, less than 20 packs a day; no comorbidities; no other treatment is taken; no vitamin supplementation at the start of treatment.

(75) TABLE-US-00008 TABLE 8 2010 WHO Before After PARAMETERS Standards treatment treatment volume >1.5 ml >1.5 ml no change pH 7.2-8 7.2-8 no change Count 15 million/ml <1 million/ml <1 million/ml Motility >40% total motility >40% total no change >32% progressive motility motility >32% progressive motility Morphology >4% according to >4% according no change Kruger to Kruger Vitality >58% >58% no change leukocytes <1 million/ml <1 million/ml no change

(76) Conclusion on the results presented in Table 8:

(77) Before treatment, the patient experiences cryptozoospermia. After treatment, cryptozoospermia (concentration less than 1 million/ml) was not improved. No adverse effects are reported. iii. Information about the patient(s) whose results are listed in Table 9 below: smoker, less than 20 packs a day; no comorbidities no other treatment is taken no vitamin supplementation at the start of treatment

(78) TABLE-US-00009 TABLE 9 2010 WHO Before After PARAMETERS Standards treatment treatment volume >1.5 ml >1.5 ml >1.5 ml pH 7.2-8 7.2-8 7.2-8 Count 15 million/ml 10 million/ml no change >39 million 40 million Motility >40% total motility 27% total 32% total >32% progressive motility motility motility Morphology >4% according to  1%  4% Kruger Vitality >58% 52% 55% leukocytes <1 million/ml <1 million/ml <1 million/ml

(79) Conclusion on the results presented in Table 9:

(80) Before treatment, the patient has a normal-low sperm count of 40 million sperm per ejaculate. Sperm concentration is low, at 10 million/ml. Sperm motility is also low, at 27%. Vitality is about 52% and the morphology is abnormal to 1% of typical forms. After treatment, there is: a 32% improvement in the motility and 55% in vitality; and an improvement of 4% in morphology of normal forms. On the other hand, the count and concentration remain unchanged. No adverse effects are reported. iv. Information about the patient(s) whose results are listed in Table 10 below: Between 20-50 years Healthy subjects with a balanced diet Exclusion of azoospermia and excretory sterility non-smoker grade 1 left varicocele (Varicocele is characterised by the dilation of a vein (varicose vein) in the spermatic cord, a fibrous cord located in the sacs above each testicle, which connects each one to the scrotum) no comorbidities no other treatment is taken no vitamin supplementation at the start of treatment

(81) TABLE-US-00010 TABLE 10 2010 WHO Before After PARAMETERS Standards treatment treatment volume >1.5 ml 1.8 ml 2.5 ml pH 7.2-8 7.2-8 8.1 Count 15 million/ml 1000/ml  0.6M/ml >39 million 15M Motility >40% total motility 7% progressive 46% total >32% progressive 28% progressive motility leukocytes <1 million/ml — 2%
In addition, the patient evaluated the product as shown in Table 11 below:

(82) TABLE-US-00011 TABLE 11 Patient satisfaction questionnaire: How do you judge the effectiveness 0 = Very effective of this product on the improvement 1 = Quite effective x of spermogram parameters? 2 = Not very effective 3 = Completely ineffective How do you judge the acceptability 0 = Very good x of this product (in terms of intake 1 = Good constraints, possible taste of the 2 = Average tablets, etc)? 3 = Bad Would you agree to use this product 1 = Yes, definitely on a regular basis? 2 = Yes, if necessary x 3 = Not at all

(83) Conclusion on the results presented in Table 10:

(84) Before treatment, the patient has a low sperm concentration at 1000/ml. Sperm motility is also low, at 7%. After treatment, there is: an increase in volume; a significant increase in sperm concentration to 0.6M/ml a large improvement in motility to 46% total and 28% progressive.

(85) Patients consider the treatment to be quite effective. No adverse effects are reported. v. Information about the patient(s) whose results are listed in Table 12 below: non-smoker Between 20-50 years Healthy subjects with a balanced diet Exclusion of azoospermia and excretory sterility no other treatment is taken no comorbidities no vitamin supplementation at the start of treatment

(86) TABLE-US-00012 TABLE 12 2010 WHO Before After PARAMETERS Standards treatment treatment volume >1.5 ml 4 ml 3 ml pH 7.2-8 7.8 7.8 Count 15 million/ml .sup. 17M/ml .sup. 40M/ml >39 million Motility >40% total motility 50% total 50% total >32% progressive 41% progressive 41% progressive motility Morphology >4% according to 2%  1% Kruger Vitality >58% 75%  79% leukocytes <1 million/ml 1%  2%
In addition, the patient evaluated the product as shown in Table 13 below:

(87) TABLE-US-00013 TABLE 13 Patient satisfaction questionnaire: How do you judge the effectiveness 0 = Very effective of this product on the improvement 1 = Quite effective x of spermogram parameters? 2 = Not very effective 3 = Completely ineffective How do you judge the acceptability 0 = Very good x of this product (in terms of intake 1 = Good constraints, possible taste of the 2 = Average tablets, etc)? 3 = Bad Would you agree to use this product 1 = Yes, definitely x on a regular basis? 2 = Yes, if necessary 3 = Not at all

(88) Conclusion on the results presented in Table 12:

(89) Prior to treatment, the patient has a low sperm concentration at 15M/ml. After treatment, there is: a significant increase in sperm concentration to 40M/ml a slight improvement in vitality to 79% is also observed. No adverse effects are reported.