NZ264068A - Blocking fertility in male mammal using neem oil - Google Patents
Blocking fertility in male mammal using neem oilInfo
- Publication number
- NZ264068A NZ264068A NZ264068A NZ26406894A NZ264068A NZ 264068 A NZ264068 A NZ 264068A NZ 264068 A NZ264068 A NZ 264068A NZ 26406894 A NZ26406894 A NZ 26406894A NZ 264068 A NZ264068 A NZ 264068A
- Authority
- NZ
- New Zealand
- Prior art keywords
- neem oil
- fertility
- vas
- male
- block
- Prior art date
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/58—Meliaceae (Chinaberry or Mahogany family), e.g. Azadirachta (neem)
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- Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Alternative & Traditional Medicine (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Medical Informatics (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Description
264068
Priority Dai<K=): ...St&b.L?.2 .3.bb.3....
Comptei» Sp^itic&iion Fifed: jaalnlaH:... Class: J<.S.Sia»:
reww
Publication Date:.... P.O. Journal No:
Patents Form No. 5
Our Ret: JB203491
NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION
HEEM COMPONENT AS A MALE CONTRACEPTIVE
We, INTERNATIONAL DEVELOPMENT RESEARCH CENTRE, a Canadian company of 250 Albert Street, Ottawa, Ontario K1G 3H9, Canada hereby declare the invention, for which we pray that a patent may be granted to us and the method by which it is to be performed, to be particularly described in and by the following statement:
PT0595353
(followed by page la)
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lo
FIELD OF THE INVENTION
The present invention relates to a method for the control of fertility in males. In particular, the invention relates to'the intra-vas administration of neem oil to the male resulting in a long term block in fertility.
BACKGROUND OF THE INVENTION
The neem tree (Azadirachta lndlca or Melia azadirachta)
found throughout India and other countries has been reported to have various medicinal utilities. In particular, various parts of the tree have been utilized in the treatment of arthritis (Pillai and Santhakuniari 1981), inflammation (Okpanyi and Ezeukwu 1981)
/
and malaria (Obbaseki and Jeged-Fadunsin, 1986). Neem oil, also known as oil of Margosa, has been shown to possess anti-diabetic (Chakrabarty and Poddar, 1984) anti-bacterial (Singh and Sastry, 1981), anti-fungal (Kher and Chaurasia, 1977 ) and ant'i-fertility effects in the female (Talwar, United States Patent No.
,196,197).
The realization of the anti-fertility effects of- neem oil in the female animal was quite important especially in view of the problems with population control in most developing countries. Alternative forms of birth control are also desirable in industrialized countries due to the recognized problems associated with known contraceptives. However, most contraceptives are aimed at controlling fertility in the female which unfairly imposes sole responsibility for birth control on the female. One accepted method of contraception in the male has been a vasectomy.
wever, this procedure has been reported to give rise to problems
[(followed by page 2)
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related to the formation of anti-sperm antibodies and associated changes in the epididymis and testes. (Herr, et al 1987; Flickinger et al 1990). Consequently, there is a real need to provide new methods for blocking fertility in the male.
SUMMARY OF THE INVENTION
t
The present ihvention relates to a new method for controlling fertility in male mammals.. In particular, the invention demonstrates that neem oil is effective in inducing a long term block of fertility in males oy a single intra-vas admlnist rat ion.
Accordingly, the present invention provides a method of blocking the fertility in a male mammal which comprises administering neem oil or, an active component thereof into the vas deferens of the male in an amount sufficient to block the fertility of said male.
The present invention also provides a male antlfertility agent comprising neem oil or an active component thereof in an amount sufficient to block fertility In a male mammal.
The block in fertility has been demonstrated to be reversible, lasting from about 5 to about 11 months after a single intra-vas injection of neem oil. .
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE It Photomicrograph of a portion of cauda epididymis of a rat 4 weeks after intra-vas administration of
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FIGURE 2« Photomicrograph of a portion of cauda epididymis of a rat, 4 weeks after intra-vas administration of neem oil. Note normal morphology of the ducts without any evidence of inflammatory reaction and the absence of sperm in the ductular lumen. X 250
FIGURE! 3« Photomicrograph of a section through vas deferens of a'rat, 4 weeks after intra-vas administration of peanut oil, showing normal morphology and the presence of sperm in the lumen. X 250
FIGURE 4« Photomicrograph of a section through vas deferens of a rat, 4 weeks after intra-vas administration of neem oil, showing normal morphology without any evidence of Inflammatory reaction and the absence of sperm in the lumen. X 250
i
FIGURE 5s Photomicrograph of a portion of testis of rat, 4 weeks after intra-vas administration of peanut oil, showing normal morphology of the seminiferous tubules . + X 100
FIGURE 6» Higher magnification of figure 5, showing seminiferous tubules. Normal spermatogenesis can be noted in the seminiferous epithelium. X 200
FIGURE It Photomicrograph of a portion of testis of rat, 4 weeks after intra-vas administration of neem oil, showing significant reduction in seminiferous tubule diameter and the arrest of spermatogenic process. X 100
FIGURE 8» Higher magnification of the same testis shown in figure 7, showing the presence of early spermatogenic cells in the seminiferous tubules. No leukocytic infiltration is seen in the interstitial space. X 200
FIGURE 91 Effect of unilateral (left side)
°A
$v' *t-1
(- ~
12 5 ncj J93^ j
264 0 68
4
administration of neem oil on the testicular weight in rats. The values are expressed as medn S.E.M. Note the significant reduction (P < .01) of the testicular weight by 6 weeks after treatment. "
FIGURE 10. Testosterone levels in rats following intra-vas treatment with neem oil showing normal profile up to 9 months '
FIGURE 11. ELISA values for anti-sperm antibody in rats, up to 9 months, following intra-vas administration of neem oil. Titres are expressed as mean optical density (OD) values at sera dilution of 1*50; Berum from a sperm-immunized male rat was used as a positive control (-C).
FIGURE 12. Lymphocyte proliferative response of regional lymph node cells to in vitro challenge with Concanavalin A (Con-A) (5 ug/ml)- fof 72 hours as measured by 3„-thymldine n
incorporation. The values are expressed as mean S.E.M.
Experimental groups I, II and III represent animals receiving saline, peanut oil and neem oil in the left vas deferens,
respectively? in all groups, right vase was left untreated. In
Group III, a significant (P < .001) increase in lymphocyte proliferative response to Con-A was noted in the lymph node cells of the treated side as compared to that of the contralateral untreated side. No significant difference was noted bet^^Sfrj'Ptafi two sides in the control groups, I and II. ff >
§ o
Iz *"
\ 25 OCT'334/
EXAMPLE 1 \* 0 J
The following example Is provided to illustratex^f§,:!:,,^ present Invention. The invention is not meant to be restricted
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thereto. The example demonstrates thay neem oil is effective in blocking fertility in male rats.
MATERIAL AND METHODS
ANIMALSi -
Malfe Wistar rats of proven fertility were used in this study. Animals were maintained under standard laboratory conditions. Water'and dry pellet diet were given ad libitum.
NEEM OILt
Neem oil was expressed from decorticated-kernels of Azadirachta indica by mechanical expeller at ambient temperature t
and centrifuged to remove particulate material. The oil was used within 3 months of extraction since it is known to loose its biological activity following longer storage and exposure to light. SURGICAL PROCEDURESt
Surgical procedures were carried out under anaesthesia (intra-peritoneal injection of 1 ml of 1% avertin: 2,2,2-tri-bromomethanol in tertiary arnyl alcohol) . The vas deferens was exposed through a midline incision on the lower abdomen? 50 ul of neem oil (or peanut oil) was administered into the lumen of the vas using a 26 gauge needle fitted to a glass syringe? Initially i
the procedure was standardized by injecting India ink into the vas. The incision was closed using autoclips. There was no mortality due to the surgical procedure and the animals revived
>
quickly.
FERTILITY STUDIESi
All animals were caged with females (It2) of proven fertility, 4 weeks after surgery. Mating studies were continued
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Vaginal smears were taken dally for monitoring sperm positivity, reproductive eyelieity and pregnancy? pregnancies were documented by delivery of pups.
_ k
SERA SAMPLESi
Blood samples were collected from orbital plexus for measurement of testosterone and anti-sperm antibody titres, before «
and at weekly intervals following the treatment. The serum was separated and stored at -20°C until assayed.
HISTOLOGYi s
Some animals were sacrificed at 2, 4 and 6 weeks after the treatment, the remaining at the end of the fertility studies.
Testis along with epididymis and vas deferens was dissected out?
♦
testicular weight was recorded and the tissue samples were immediately fixed in a solution containing 4% paraformaldehyde and 2.5% glutarsldehyde in 0.1 M cacodylate buffer for 4-6 hours at 4°C. Samples were post-fixed in 1% osmium tetroxide, dehydrated in graded series of ethanol and embedded in epon-araldite. Semlthin serial sections, stained with toluidine blue, were studied under a Zeiss Axiophot photomicroscope.
TESTOSTERONE ASSAY»
Testosterone levels in individual serum samples were determined according to the method described by Brenner et eN7 o
(1973).
fl z
ANTI-SPERM ANTIBODY ASSAY. I 25 OCT
Anti-sperm antibody tit re in the serum samples wa^£c^\v^ determined by ELISA as described by Herr et al (1986). Serum from a sperm-immunized rat was used as positive control. All sera were diluted 1<10, It50 and It 100 for the assay, and the results were
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plotted as mean OD values at 1*50 dilution for all samples.
*
LYMPHOCYTE PROLIFERATION A&5AY:
Para-aortic and inguinal lymph node cells from experimental and control groupg were isolated, washed and suspended in RPMI containing 10% fetal calf serum at a concentration of 5 X 106 cells/ml. One hundred microliters of this suspension were plated in 96-well plktes and cultured for 72 hours with 100 ul of Con A (5 ug/ml)? controls without Con A were maintained. 3H-Thymidine (1 uCi) was added to each well and the cells were" harvested after 18 hours? thymidine incorporation was measured using a beta counter (Phamacia). All tests were done in triplicate.
RESULTS
A group of 10 animals were given 50 ul of neem oil in the vas deferens of each side? a control group of equal numbers received the same volume of peanut oil on both sides. Animals were put on continuous mating with females of proven fertility, 4 weeks after the treatment. The results showed that administration of peanut oil in the vas.did not affect fertility, all animals of this control group could impregnate their female partners. 'The animals treated with neem oil, however, remained infertile during the 8 month period of observation? none of the females caged with neem oil treated males showed sperm positive mating. The treatment, however, did not affect libido and mating since the females frequently manifested pseudo-pregnancy for 10 - 12 days as evidenced by prolonged diestrous stage. The control animals impregnated females within first or second cycle.
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8
following application of neem oil (Pigs. 1 - 4). The lumen of excurrent ducts was, however, devoid of spermatozoa by 4 weeks of treatment (Figs. 2 & 4). No occlusion, granuloma or inflammatory changes were noted in the vas and epididymis; the lumen was patent and the epithelial cells lining the ducts had normal morphology {Figs. 2 & 4) .
Histological studies on testes revealed that by intra-vas application of neem oil, spermatogenesis was impaired as early as two weeks after the treatment. By 4 weeks, a drastic reduction of seminiferous tubule diameter and complete block in spermatogenesis was noted (Figs. 5-8); the seminiferous tubules contained mainly spermatogonia and primary spermatocytes (Figs. 7
»
& 8). The Leydig cells, however, appeared normal. There was no leukocytic infiltration in the testicular lnterstitlum. The block of spermatogenesis was observed even after 9 months of treatment; the seminiferous tubules, however, did not show any sign of degeneration or resorption and still contained a few spermatogonia.
Unilateral administration of neem oil in the vas Jiad. a
*
similar effect on testis but only on the side of treatment. While the testicular weight of the control side remained unaffected, the treated side showed a significant reduction (P < .01) in gross weight by 6 weeks of treatment (Fig. 9). The block in spermatogenesis was also noted only on the treated side.
Serum testosterone levels, as shown in Fig. 10, indicated that the bilateral intra-vas administration of neem oil did not affect the endocrine functions of the testes? the testosterone levels were comparable to that in control animals. No anti-sperm antibodies could be detected in serum of ueem oil treat
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9
(Fig. 11) .'
The effect of intra-vas administration of neem oil on the regional draining lymph nodes (para-aortic and inguinal) wais
»
studied in order to understand the localized effect following unilateral treatment. Three experimental groups of six animals each were given saline, peanut oil and neem oil, respectively,
into the left' vas; in all groups, the right vas, was left untreated. The lymph nodes of both sides were removed after 2 weeks and were challenged in vitro with Con-A (5 ug/ml). The results, shown t
in Figure 12, indicate that in Group III, the lymph node cells of the treated side show significantly higher response (P < .001) to Con-A as compared to that of the contralateral side? the two control groups (I and II) had no difference between the'response of left and right side.
DISCUSSION
This example demonstrates that a single administration of neem oil in the vas induces a long term block of fertility of male rats. The treatment does not lead to any inflammatory reaction or occlusion in the vas or epididymis; in fact, In Indian traditional medicine,, neem oil Is known for its antiinflammatory properties and this has been confirmed by recent experimental studies (Labadie et al, 1989). The long term anti-fertility effect as noted in the present study appears to be due to the arrest of spermatogenesis. It is possible that the block of fertility may be reversible since the spermatogenic stem cells, the spermatogonia, were still present in the seminiferous tubules after 9 months of treatment. Intra-vas treatment with neem oil did not affect testosterone production or libido. The
264068
continued to mate normally as evidenced by induction of pseudo-pregnancy in females for 10 to 12 days, a phenomenon well known to occur in rodents after infertile mating. The treatment did not lead to formation of anti-sperm antibodies. The block of spermatogenesis does not seem to be mediated by any systemic mechanism since the unilateral administration of neem oil affected only the testds of that side? a systemic involvement would have affected testes of both sides.
ing a single application of neem oil in the vas may possibly be t '
mediated'by local cell mediated immune mechanisms. This study demonstrates that unilateral intra-vas application of neem oil activates the immune cell population in the draining lymph nodes of the treated side, which show enhanced response to mitogenlc challenge, whereas the lymph nodes of untreated contralateral aide show normal response. The Inventors have earlier demonstrated that neem oil possesses immuno-stimulatory properties and that it non-specifically activates the immunocompetant cells. In i
particular, it enhances the phagocytic and antigen presenting ability of the macrophages (Upadhyay et al, 1992). Neem oil also induces production of gamma interferon (Upadhyay et al, 1992), an important mediator of cellular immune responses. Intra-vas administration of neem oil may activate the immuno-competent cells in the regional draining lymph nodes. Recent reports suggest that the cytokines produced locally may be involved in the regulation of spermatogenic process (Pollanen et al, 1990). Although the precise role of these cytokines in the regulation of spermatogenesis is not clear, intra-vas
The mechanism of long term spermatogenic arrest follow-
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may possibly Interfere.with normal production of the Immune cell products necessary for the maintenance of the spermatogenic process.
The intra-vas application of neem oil for inducing long term block of fertility, without loss of androgens and libido, could serve as an alternate to vasectomy. Although vasectomy has i
been widely accepted for male contraception, it does give rise to problems related to formation of anti-sperm antibodies and t •
associated inflammatory changes in the epididymis and testes (Herr et al, 1987; Flickinger et al, 1990). The procedure for administration of neem oil In the vas may be relatively easier and less traumatic than ligation and incision of the vas. The vas deferens is a convenient site for administration. Non-surgical techniques for intra-vas injections have been worked out and are practised in some countries such as China and India. The anti-inflammatory and anti-microbial properties of neem oil may be i
an added advantage during post-surgical recovery.
While the above example describes the ability of neem oil to block fertility in the male rat, it would be reasonable to predict that the same effect would be observed in other mammals such as humans and monkeys. Experiments are currently underway in the monkey using a dose of 0.2 to 0.5 ml. It is predicted that an effective dose in humans would be approximately up to 2 ml. It is also predicted that the anti-fertility effect would be reversible in all mammals. <
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REFERENCE S
Brenner PF, Guerrero R, Cekan Z, Diczfalusy E.
"Radioimmunassay method for sex steroids In human plasma."
Steroids (1973) 22: 775-794.
Chakrabarty T, Poddar G. Herbal drugs in diabetes. Part I.
t
"Hypoglycaemlc activity of indigeneous plants in sterptozoticin induced diabetic rats". J. Inst. Chem.., (1984) 56» 20-22.
Flickinger CF, Herr JC, Caloras D, Sisak JR, Howards SS. "Inflammatory changes in the epididymis after vasectomy in the Lewis rat." Bio Reprod (1990) 43: 34-45.
i
Herr JC, Flickinger CJ, Howards SS, Yarbro S, Spell DR, Caloras D, Gallien TN. "An enzyme-linked immunosorbent assay for measuring i *
antisperm autoantibodies following vasectomy in Lewis rats." Am J Reprod Immunol Microbiol (1986) 11.» 75-81.
Herr JC, Flickinger CJ, Howards SS, Caloras D, Yarbro ES, Spell DR, Gallien TN. "The relation between antisperm antibodies and testicular alternations after vasectomy and vasovasotomy in Lewis rats." Bio Reprod (1987) 37: 1297-1305.
»
Kher A, Chaurasia SC. "Anti-fungal activity of essential oils of three medicinal plants Indian Drugs" (1977) 15:41-42.
>
Labadle RP, Van der Nat JM, Simons JM, Kro.es BH, Van der Berg AJJ, Hart LA, Van der Sluis WG. "Advances in immunomodulsting
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from medicinal plants and traditional preparations." Proc Sixth Asian Symp Med Plants and Spices, Bandung, Indonesia. (1989) 408-428.
" %
Obaseki AO, Jegede-Fadunsin H.A. "Anti-malarial activity of Azadirachta indica". Flloterapla, (1986) 57: £47-251.
t
Okapanyi SN, Ezeukwu G.C."Anti-inflamatory and anti-pyretic activities of Axaridachta India". Planta Med., (1981) 41134-39.
Pillai MR, Santhakumari G. "Anti-arthritic and anti-inflamatory actions or nlmbidin". Planta Med., (1981a) 43t 59-63.
Pollanen P, yon Euler M, Soder O, 1990. "Testicular immunoregulatory factors." J Reprod Immunol (1990) .18: 51-76.
Singh N. Sastry MS, "Ant i-rnicrobia activity of neem oil.'" Indian J. Pharmacol., (1981) 13:102.
.Sinha KC, Riar SS, Tiwary RS, Dhawan AK, Bardhan J, Thomas P, Kain AK, Jain RK. "Neem oil as a vaginal contraceptive." Indian J Med Res (1984) 79, 131-135.
i
Upadhyay SN, Dhawan S, Garg S, Talwar GP. "Immunomodulatory effects of neem (Azadirachta indica) oil." Int J Immunopharmacol. (1992) 14:1187-1193. .
Claims (10)
1. A method of blocking the fertility In a male mammal which comprises administering neem oil or an active component thereof into the vas deferens of the male in an amount sufficient to block the fertility of said male.
2. A method according to claim 1 wherein said block in fertility is reversible.
3. A method according to claim 1 wherein said neem oil or active component thereof is administered in an amount sufficient to block fertillty for about 5 to about 11 months.
4. A method according to claim 3 wherein said block in fertility is for about 8 months. i
5. A method according to claim 1 wherein said male mammal is a human. i •
6. A method according to claim 5 wherein the neem oil is administered in a dosage of up to 2 milliliters.
7. A method according to claim 1 wherein said neem oil or active component thereof is administered as a single dose.
8. A male antifertility agent comprising neem oil or an active component thereof in an amount sufficient to block fertility in a male mammal. 264068 15
9. A method according to any one of the preceding claims, substantially as herein described.
10. A method according to claim 1, substantially as described with reference to any one of the accompanying drawings or any one of the Examples. INTERNATIONAL DEVELOPMENT RESEARCH CENTRE fBy Its Attorneys BALDWIN. SON & CAREY
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
IN766DE1993 | 1993-07-22 | ||
US08/115,867 US5501855A (en) | 1993-09-02 | 1993-09-02 | Neem oil as a male contraceptive |
Publications (1)
Publication Number | Publication Date |
---|---|
NZ264068A true NZ264068A (en) | 1996-09-25 |
Family
ID=26324666
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
NZ264068A NZ264068A (en) | 1993-07-22 | 1994-07-22 | Blocking fertility in male mammal using neem oil |
Country Status (5)
Country | Link |
---|---|
AU (1) | AU6866794A (en) |
CA (1) | CA2128649A1 (en) |
GB (1) | GB2281511A (en) |
IE (1) | IE940592A1 (en) |
NZ (1) | NZ264068A (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0930073B1 (en) | 1998-01-14 | 2004-08-04 | National Research Development Corporation of India | Process for preparation of spermicidal agent |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5196197A (en) * | 1990-08-29 | 1993-03-23 | National Institute Of Immunology | Reversible fertility control for prevention of pregnancy in females |
-
1994
- 1994-07-22 IE IE940592A patent/IE940592A1/en not_active IP Right Cessation
- 1994-07-22 AU AU68667/94A patent/AU6866794A/en not_active Abandoned
- 1994-07-22 CA CA002128649A patent/CA2128649A1/en not_active Abandoned
- 1994-07-22 NZ NZ264068A patent/NZ264068A/en unknown
- 1994-07-22 GB GB9414835A patent/GB2281511A/en not_active Withdrawn
Also Published As
Publication number | Publication date |
---|---|
CA2128649A1 (en) | 1995-01-23 |
IE940592A1 (en) | 1995-01-25 |
AU6866794A (en) | 1995-02-02 |
GB9414835D0 (en) | 1994-09-14 |
GB2281511A (en) | 1995-03-08 |
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