Potentiation of the gonadotoxicity of Cytoxan in the dog by adjuvant treatment with a luteinizing hormone-releasing hormone agonist.

This study evaluates the effect on spermatogenesis of coadministration of Cytoxan (cyclophosphamide) and nafarelin, a luteinizing hormone-releasing hormone agonist. Nafarelin causes complete aspermatogenesis in dogs by interrupting the hypothalamic-pituitary-gonadal axis, which might protect against the testicular cytotoxicity associated with cyclophosphamide. The four treatment groups, each consisting of 2 mature male beagle dogs, were (a) no drug; (b) cyclophosphamide (p.o. 3x weekly for 43 and 48 wk for a total dose of 582 and 709 mg/kg, with dose varying according to weekly hematological profile); (c) nafarelin (2 micrograms/kg s.c. daily for 48 and 52 wk); and (d) cyclophosphamide plus nafarelin [same schedule as above with cyclophosphamide (570 and 698 mg/kg total dose) starting 7 wk after beginning nafarelin]. Plasma testosterone, spermatogenesis, and ejaculate volume were completely suppressed by nafarelin prior to starting cyclophosphamide. By 2 wk after cessation of treatment (posttreatment, PT), plasma testosterone reached normal levels, and at 5 wk PT ejaculates appeared which reached normal volumes 2 to 3 wk later. Normally motile ejaculated spermatozoa were noted at 6 to 8 wk PT in nafarelin-only-treated animals; normal sperm numbers were reached at 14 wk PT. The animals receiving cyclophosphamide plus nafarelin were azoospermic for the entire 65-wk PT period, and at 65 wk PT no germinal cells were found upon evaluation of testicular histology. Sperm numbers in cyclophosphamide-only-treated animals began to rise 10-11 wk PT and reached 150 x 10(6) sperm/ejaculate at approximately 65 wk PT (contemporaneous control dogs had sperm numbers of approximately 300-600 x 10(6)/ejaculate). Spermatogenesis in these cyclophosphamide-only-treated animals was normal in most seminiferous tubules at this time. The addition of nafarelin to cyclophosphamide treatment thus exacerbated the deleterious effects of cyclophosphamide on the testes, suggesting caution for use of such a protocol clinically.

Toxicity and tolerability of recombinant human ciliary neurotrophic factor in patients with amyotrophic lateral sclerosis.

We examined the toxicity of both single and multiple subcutaneous injections of recombinant human ciliary neurotrophic factor (rhCNTF) in 72 patients with ALS, in doses ranging from 2 to 100 micrograms/kg. Adverse events were generally dose related and ranged from mild to severe. The tolerability of daily subcutaneous rhCNTF was equivalent to placebo at doses < or = 5 micrograms/kg/day. At higher doses, anorexia, weight loss, reactivation of herpes simplex virus (HSV1) labialis/stomatitis, cough, and increased oral secretions occurred.

Potent gonadotropin releasing hormone antagonists with low histamine-releasing activity.

The incorporation of Arg residues into position 6 of gonadotropin releasing hormone antagonists had resulted in compounds with increased in vivo potency but also made these analogues potent mast cell degranulators. We have focused on the substitution of position 8 by hArg(R)2 (NG,NG'-dialkylhomoarginine) substitutions, based on the hypotheses that the Arg-Pro sequence is of major importance for this side effect and that shielding of the charge may be an effective way to block degranulation. Analogues in four series were evaluated: (A) [N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Pal-(3)3,6,Arg5,hArg(R)2(8),D-+ ++Ala10]GnRH, (B) [N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Pal(3)3,6,hArg(R)2(5,8),D-Ala10 ]-GnRH, (C) [N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Pal(3)3,6,hArg(R)2(8),D-Ala10]G nRH, (D) [N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Pal(3)3,D-hArg(R)2(6),hArg(R)2( 8),D-Ala10]GnRH. Although substitution by hArg(Et)2, hArg(Bu), hArg(CH2)3, and hArg(CH2CF3)2 was tested, in each series the hArg(Et)2 residue was superior. Two compounds were considered for clinical evaluation: [N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Pal(3)3,6,hArg(Et)2(8),D-Ala10] GnRH and [N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Pal(3)3,D-hArg(Et)2(6),hArg(Et) 2(8),D- Ala10]GnRH (ganirelix acetate). These compounds had high potency for ovulation suppression and low histamine-releasing potency in vitro (ED50 = 0.6, 0.29 microgram/rat and EC50 = 196, 13 micrograms/mL, respectively). Ganirelix is currently in Phase II clinical trials and appears to be the most potent GnRH antagonist tested in humans (based upon ED50 for 24-h suppression of testosterone levels).

Clinical improvements of specific seminal deficiencies via intercourse with a seminal collection device versus masturbation.

In the present study of 50 patients, it was determined that those with seminal deficiencies in masturbated samples showed greater improvement in semen parameters with I-SCD use than the nondeficient groups with which they were compared. The use of I-SCD in oligospermia and OTA Syndrome is therefore indicated, both diagnostically and therapeutically. The subjective rating of sexual stimulation elicited when I-SCD was used was far superior than with masturbation. It is believed the success of I-SCD is due, in part, to this greater degree of sexual stimulation, presumably by added loading of the vas deferens prior to ejaculation.

Evaluation of the in vivo efficacy of a new vaginal contraceptive agent in stumptailed macaques.

The effect of precoital vaginal placement of three spermicidal formulations on postcoitally recovered vaginal and cervical spermatozoa was compared. Periovulatory stumptailed macaque monkeys were administered intravaginally 1 ml of Ramses Contraceptive Vaginal Jelly (C. Schmid Products Company, Little Fall, NJ), or a pluronic gel formulation containing 0, 0.1, or 1.0% of a new spermicidal compound, RS-37367. All spermicide-containing formulations reduced the motility of both vaginal and cervical sperm, compared with the gel containing no spermicide. The 1.0% RS-37367 formulation was particularly effective. No motile spermatozoa were recovered from either location, and only 0.5% of the numbers of cervical spermatozoa recovered in control animals were recoverable from this treatment group (P less than 0.0003). The formulation containing 0.1% RS-37367 appeared to be bioequivalent to the Ramses formulation containing 5% nonoxynol-9. These in vivo results support previous in vitro comparisons in which RS-37367 was estimated to be 50 times as potent a spermatostatic agent as nonoxynol-9.

Assessment of a new spermicidal agent against ejaculated dog and human spermatozoa in vitro.

The spermatostatic potencies of a new vaginal contraceptive agent, RS-37367, and a standard surfactant compound, nonoxynol-9, have been compared by using ejaculated dog and human spermatozoa. RS-37367 was 25 to 50 times more potent than nonoxynol-9 against dog spermatozoa. Nonparallel concentration-response lines were obtained against human spermatozoa. Concentrations of RS-37367 causing immediate spermatostasis against dog spermatozoa resulted in vesiculation of the plasma and outer acrosomal membranes of spermatozoa; similarly, immediately spermatostatic concentrations of nonoxynol-9 were associated with the previously documented generalized membrane stripping. The activities of both RS-37367 and nonoxynol-9 were affected by the concentration of dog spermatozoa in semen-compound mixtures. Short-term (5-minute) exposure of spermatozoa to concentrations of RS-37367 not immediately spermatostatic resulted in progressive immobilization of spermatozoa. Extensive washing of the spermatozoa was not able to reverse this effect, in contrast to spermatozoa transiently exposed to nonoxynol-9.

Relationship of human sperm acrosin and proacrosin to semen parameters. II. Correlations.

Previous results, simultaneously confirmed by others, suggest that a relationship exists between sperm acrosin levels and fertility in man. The assessment of sperm acrosin may therefore be a useful addition to the semen analysis, but only if the more standard semen parameter measurements cannot predict acrosin levels. Ejaculates from 102 men were analyzed and the relationship of the sperm acrosin system (acrosin, proacrosin, and acrosin inhibitor) to other seminal characteristics was determined. Very little correlation was observed between enzymatic and nonenzymatic parameters. Only five non-enzymatic parameters, all of which were morphologic, showed correlation coefficients of greater than or equal to 0.35 with acrosin and proacrosin, but none had an r-value above 0.48. The total acrosin and proacrosin levels were highly correlated to each other (r = 0.93). It is concluded that sperm acrosin/proacrosin levels cannot be predicted by other seminal parameters; thus, measurement of sperm acrosin/proacrosin may be clinically useful as a diagnostic parameters.

Same day appearance of orally administered, spermicidal 1-substituted imidazoles in dog ejaculates.

Within hours after administration of high oral doses of ketoconazole to males of various species, the intact compound appears in the seminal plasma, leading to immobilization of spermatozoa in ejaculates collected several hours later. The present report describes in vitro and in vivo characterization studies of several new compounds identified from a series of 1-substituted imidazole compounds. Relative rank order of in vitro potencies of the four compounds studied was RS-29984 greater than RS-90847 greater than RS-41353 greater than RS-68287. Oral administration of single doses of these compounds ranging between 10 and 95 mg/kg, followed by ejaculation of the animals at various times after dosing, showed that their relative potencies for decreasing sperm motility were RS-41353 greater than RS-68287 = RS-90847 greater than RS-29984. Four hours after animals were given 30 mg/kg of RS-41353, spermatozoa in the ejaculates had zero forward progression within 30 to 40 minutes after the start of ejaculation. A preliminary metabolic study indicated that the apparently greater potency of RS-68287 in vivo than in vitro was probably not due to metabolic activation. The androgen-suppressing activity of RS-29984 and RS-90847 was shown to be less than that of ketoconazole. These data indicate that orally active inhibitors of sperm motility that exert their effects after ejaculation may be feasible, and suggest that this novel approach to male contraception warrants further investigation.

Development and evaluation of an inhibitor-releasing matrix for intrauterine devices.

Antifibrinolytic agents when released into the uterine cavity decrease menorrhagia associated with IUD use. Our objective was to develop a matrix that could be incorporated onto an IUD and release anti-fibrinolytic agents. The copolymer ethylene-vinyl acetate (EVA) was selected for detailed study because it has the advantage over other materials in that it can release large molecular weight substances for more than 100 days, and allows incorporation of large amounts of anti-fibrinolytic agents with different molecular weights. Two compounds, tranexamic acid (AMCA, MW=157) and Trasylol (Kunitz pancreatic trypsin inhibitor, (MW=6,500) were incorporated into the EVA matrix and their release rates measured. In vitro studies with AMCA showed that after the initial burst, a constant high release rate was obtained over a prolonged period of time. The in utero release rate of AMCA from the EVA matrix in rabbits was similar to that obtained in vitro. By contrast, the release rate of Trasylol decreased to low levels during incubation in vitro. The release rate of Trasylol in utero however, appeared to be higher than that in vitro.

Gonadotropin-releasing hormone agonist analog (nafarelin): a useful diagnostic agent for the distinction of constitutional growth delay from hypogonadotropic hypogonadism.

To determine the usefulness of a GnRH agonist analog as a diagnostic test to distinguish between constitutional delay of growth (CGD) in boys with Tanner stage I of sexual development and patients with hypogonadotropic hypogonadism (HH), we evaluated six boys (mean age 15 yr 4 m) and five HH patients (mean age 20 yr 4 m). In addition, 20 normal healthy men aged 21 yr to 50 yr received either nafarelin or GnRH followed two weeks later by the other test in order to compare the efficacy of each of these tests and to evaluate the optimal sampling times for the nafarelin test. All subjects were healthy, and had not received hormonal replacement for at least 2 months prior to enrollment in the study. Each man had four baseline blood samples before and at timed intervals following the administration of either GnRH or nafarelin. Each of the patients had blood withdrawn every 15 min during 12 h overnight followed by a single s.c. injection of nafarelin (1 microgram(s)/kg up to 100 microgram(s)), except two HH patients who did not have an overnight study. Blood samples were obtained at timed intervals for 24 h. LH, FSH, T and E2 were measured by RIA. Baseline concentrations of plasma LH, FSH and T were similar before the administration of either GnRH or nafarelin in the group of normal men. Peak stimulation of plasma LH, FSH and T released by nafarelin was significantly higher, and it took a longer time to reach the peak maximum, than after GnRH (p < 0.001). Mean nocturnal LH was 5.5 +/- 0.9 IU/I for the CGD group, and 2.7 +/- 0.7 IU/I for HH (p < 0.02). Mean nocturnal FSH was 5.1 +/- 1.0 and 2.5 +/- 0.2 IU/I whereas mean nocturnal T concentrations were 4.2 +/- 0.8 and 0.7 +/- 0.2 nmol/I (CGD vs HH, respectively, p < 0.02). Peak LH responses to nafarelin were 36.9 +/- 8.9 IU/I for the CGD group, and 7.0 +/- 2.0 IU/I for the HH group (p < 0.001). Peak FSH released by nafarelin was 14.2 +/- 2.4 IU/I for the CGD group and 4.8 +/- 2.0 IU/I for the HH group (p < 0.02). Peak T was reached 24 h following nafarelin injection and was 5.7 +/- 1.7 nmol/I for the CGD group and 0.3 +/- 0.2 nmol/I for the HH group (p < 0.001). The results obtained indicate that in early stages of puberty (before detectable changes of sexual maturation) the nafarelin test, with measurements of LH, FSH and T in blood or in urine, is superior to and more practical than overnight hormonal estimates to clearly distinguish CGD from HH.

Quantitative analysis of blood and fat in suction lipectomy aspirates.

This study of suction lipectomy aspirates from 15 consecutive patients was undertaken to biochemically quantitate the blood-to-fat ratios of the aspirates. A wide variation in the blood-to-fat ratios (8 to 54 percent) was noted, but the authors failed to demonstrate any relationship between the blood-to-fat ratios and the suction lipectomy operative site. Prophylactic measures to allow treatment of patients in a consistently safe manner include carefully screening of patients to exclude those with bleeding disorders or significant illnesses, perioperative oral iron therapy, infiltrating the operation site with a dilute epinephrine solution, hydrating the patients adequately perioperatively, using smaller-diameter cannulas for the aspiration, minimizing aspiration once the aspirate turns grossly bloody, and limiting the aspirate to a volume of less than 1750 ml for any operative procedure.

Acrosin of mouse spermatozoa.

Mouse spermatozoa possess a neutral proteinase, acrosin, that is to a large extent (70-80%) present in the zymogen (proacrosin) form. Acid extraction yields higher amounts of acrosin than detergent extraction. Synthetic inhibitor studies indicate that mouse acrosin has a serine and histidine at its active site and hydrolyzes the peptide bonds of lysine and arginine but of not phenylalanine. An inhibitor of acrosin is associated with mouse spermatozoa, capable of preventing the activity of at least 60% of all available acrosin. Acrosin activity is essential for fertilization because natural and synthetic inhibitors of mouse acrosin prevent the union of the gametes. Also, the relative inhibitory activity of synthetic agents toward acrosin runs approximately parallel to their antifertility activity. The percent of acrosin in the proacrosin form does not change after capacitating mouse spermatozoa in vitro.

Long-term evaluation of the effect of catheter materials on urethral tissue in dogs.

A surgical procedure was developed which allowed for the long-term evaluation of the effect of candidate Foley catheter materials on urethral tissue. The dog provided a satisfactory animal model since, after being subjected to the described procedure, this animal remained continent and free of urinary tract infection. Also, each animal served as its own control by examining tissue from a portion of the urethra which was not in contact with the implanted material.

Polyamine inhibition of the conversion of human proacrosin to acrosin.

The conversion of human proacrosin to acrosin was inhibited by polyamines. The order of effectiveness was spermine greater than spermidine greater than cadaverine greater than putrescine greater than 1,3,-diaminopropane. These results are similar to those obtained for the conversion of boar proacrosin to acrosin. Unlike the effects on boar acrosin, however, polyamines did not affect the esterolytic activity of human acrosin but had a slight stimulatory effect on the proteolytic activity of human acrosin.

Morphology of Fallopian tubes removed from a patient after failure of clip sterilization.

A bilateral salpingectomy was performed at the time of vaginal hysterectomy in a pregnant patient sterilized 20 months earlier by application of Bleier clips to the fallopian tubes. Clip application to the left tube had been incomplete. Undisturbed tissue (left tube) and clipped tissue (right tube) were examined and compared by light and scanning electron microscopy. The segment of tube fully grasped by the clip had a completely detached lining epithelium with coarse and flattened mucosa, a loose stroma, and disorganized muscular bundles. The left tubal mucosa was normal, whereas the right tube was stenosed at the site of clip placement, with rigid walls and a 0.175 mm luminal diameter. With progressively greater stenosis toward the clip site, mucosal destruction increased. Polypoidal mucosal folds were seen, as well as fibrous adhesions between mucosal folds. These observations indicate that tissue damage is extensive enough after clip application to require excision of the damaged segment of tube and microsurgical re-anastomosis for reversal of sterilization.

PIP: A 37-year-old woman had a bilateral salpingectomy performed at the time of vaginal hysterectomy although she had been sterilized 20 months before via application of Bleier clips to the fallopian tubes. The woman presented pregnant. Using both a scanning electron and a light microscope, undisturbed tissue (left tube where clip application had been incomplete) and clipped tissue were examined. The right tube (clipped) showed completely detached lining epithelium with coarse and flattened mucosa, loose stroms, and disorganized muscular bundles in the segment where the clip had been attached. The left tubal mucosa, where the clip had failed to attach, was perfectly normal, whereas the right tube was stenosed at the site of clip placement. The tissue destruction seen after clip sterilization is so extensive that excision of damaged segment followed by microsurgical reanastomosis seems the only procedure to reverse sterilization of this type.

Effects of various conditions of semen storage on the acrosin system of human spermatozoa.

Stability of the human sperm acrosin system (major components: non-zymogen acrosin, proacrosin and acrosin inhibitor) was studied under various conditions of semen storage used clinically or in the laboratory. Freezing at -196 degrees C caused a profound decrease in total acrosin content and in the amount of this enzyme present in zymogen form (proacrosin), but resulted in some increase in non-zymogen acrosin. Acrosin inhibitor did not appear to be significantly affected by this treatment. No relationship was present between the decreases in sperm motility induced by freezing to -196 degrees C and the alterations in total acrosin, proacrosin and non-zymogen acrosin. Storage of whole semen at -20 degrees C had deleterious effects on all the components of the acrosin system measured except for non-zymogen acrosin. Major decreases in the total acrosin, proacrosin and acrosin inhibitor occurred after only 1 day at -20 degrees C and continued slowly thereafter. Whole semen kept at room temperature for up to 24 h after ejaculation did not show any significant changes in the sperm acrosin system. Seminal plasma did not have a detrimental or stabilizing effect of acrosin and proacrosin when spermatozoa were kept at room temperature. However, removal of seminal plasma and re-suspension of spermatozoa in 0.9% NaCl resulted n the liberation of a significant amount of the acrosin inhibitor from the spermatozoa and the apparent activation of some of the proacrosin to acrosin.

Properties of a highly purified antifertility factor from human seminal plasma.

A high molecular weight antifertility factor (AF-1) was obtained in a high degree of purity from human seminal plasma by ultracentrifugation, CM-cellulose and concanavalin A chromatography, and Sepharose 6B gel filtration. A final purification step involving preparative disc electrophoresis was occasionally required. AF-1 showed a dose-dependent inhibition of the in vitro fertilizing ability of capacitated mouse spermatozoa, causing 50% inhibition of fertilization at approximately 27.5 micrograms/10(6) spermatozoa. Removal of the follicle cell layer of the oocyte did not decrease the antifertility effect of AF-1 but inhibition of fertilization was no longer observed after dispersal of the zona pellucida. The effect of AF-1 was on the spermatozoa and not on the oocytes. These results show that AF-1 treatment prevents capacitated spermatozoa from penetrating the zona pellucida and possibly the follicle cell layer. The mechanism by which AF-1 does so is not known because AF-1 did not prevent the in vitro acrosome reaction of guinea pig spermatozoa, nor did it inhibit the activity of human acrosin or bovine testicular hyaluronidase. The antifertility activity of AF-1 is reversed after recapacitation of the AF-1 treated spermatozoa and it can be assumed that AF-1 is either dissipated or loses activity during the transport of the spermatozoa through the female genital tract when capacitation takes place. AF-1 is heat labile. The properties of AF-1 are the same as those found previously for the pellet obtained by high-speed centrifugation of human seminal plasma, indicating that this is the primary, nonparticulate, high molecular weight factor with antifertility activity in human semen.

Orally administered ketoconazole rapidly appears in seminal plasma and suppresses sperm motility.

Ketoconazole has been shown to exert spermatostatic effects in vitro on ejaculated dog, monkey, and human spermatozoa. Oral administration of the compound to adult male beagle dogs (50-246 mg/kg) or rhesus monkeys (85-100 mg/kg) was associated with a decline in motility of sperm in ejaculates obtained after dosing. In dogs the decline in sperm motility was correlated with the presence of ketoconazole in the seminal plasma, although the measured concentrations of ketoconazole were no more than one tenth that needed for in vitro activity. The serum levels of testosterone in the dogs receiving oral ketoconazole were profoundly suppressed but the extreme rapidity of onset of the ex vivo effect on sperm motility, which was noted within 4 hours of dosing, makes it unlikely that testosterone withdrawal plays more than a minor role in the spermatostasis. The results in animals invite further pursuit of this novel, rapid onset, reversible, single dose use of spermatostatic agents for their potential as male contraceptives.