Factors influencing nonoxynol-9 permeation and bioactivity in cervical mucus.

The effects of delivery gel pH and osmolarity on both the mass transport and 'biodiffusion' of the spermicide nonoxynol-9 (N9) in bovine cervical mucus were evaluated. Delivery gels were calcium chloride crosslinked alginate containing 3% N9, and were manufactured over a pH range of 3.4 to 5.9 and an osmolarity range of 300 to 900 mosmol. Mass transfer parameters (diffusion coefficients and total drug loading) were determined using a new UV spectrophotometric technique while biodiffusion (the diffusion distance into mucus at which sperm are killed) was assessed using the Double Ended Test. It was found that delivery gel pH had a significant effect on spermicidal efficacy of the alginate-N9 system; biodiffusion increased with decreasing pH. Actual N9 diffusion into mucus was found to be influenced by both the delivery gel pH and osmolarity. At high N9 concentration (near the gel/mucus interface), mass transport tended to decrease with decreasing pH at the highest osmolarity. At low concentration, mass transport tended to decrease with increasing osmolarity and decrease with increasing pH at the highest osmolarity. The difference between low and high concentration behavior can be attributed to N9 micelle formation. These findings are interpreted in the context of the design of intravaginal drug delivery vehicles for spermicides.

Spectrophotometric analysis of molecular transport in gels.

An automated spectrophotometric method has been developed for analyzing molecular transport out from and into gels. A Beckman DU7500 diode-array UV-visible spectrophotometer with gel scanner was modified to accept and longitudinally scan a quartz diffusion cell, 0.3x10x40 mm. Molecules of interest are identified and concentrations quantitated via analysis of spectrophotometric absorbance peaks relative to background absorbance of the gel. Thus, concentration profiles are obtained as functions of both position and time. Test data are fitted to a diffusion model via nonlinear least-squares regression. Precision and accuracy of the method were assessed via analysis of several test molecules and gels: (1) 30 mg/ml nonoxynol-9 (N9), contained in 1% sodium alginate gel cross-linked with 2.5 mM calcium chloride, permeating standardized, reconstituted bovine cervical mucus (BCM); (2) 2.5 mg/ml sodium fluorescein, contained in and permeating 10 mg/ml and 100 mg/ml gelatin gels; and (3) 1.0 mg/ml sodium ganciclovir, contained in and permeating 10 mg/ml sodium hyaluronate gel. Diffusion coefficients for (1) and (3) were 3.8x10-7 and 54.1x10-7 cm2/s, respectively. All measurements of diffusion coefficients, partition coefficients, and solute loads obtained in this study were highly repeatable (most C.V.'s<8%). The mean diffusion coefficient for (2) was within 3% of values predicted from theory for the 100 mg/ml gel; the mean partition coefficient for (3) was within 2% of the expected value. This new technique is simpler than many traditional ones in that it does not require labeling of test molecules nor changes in refractive index of target materials. It is particularly well-suited to situations in which the target material is a gel, because no stirring of the target is necessary.

Alteration of human sperm kinematics in cervical mucus due to nonoxynol-9.

Traditional endpoints of the double-ended test (DET), a contraceptive screening assay used to evaluate the ability of a compound to permeate cervical mucus and inhibit sperm progression, ignore important information about sublethal effects upon sperm cells. Improved contraceptive agents may capitalize on such sublethal aspects. This study utilized a DET testing protocol that included measurement of human sperm motion characteristics as an indicator of cell function within spermicide-exposed human mucus. The currently available spermicide nonoxynol-9 (N9) was used as the test compound and was dissolved in two different delivery solutions, deionized (DI) water and saline, to evaluate the effects of the osmolarity and pH of the delivery vehicle on test results. The N9-water treatment demonstrated significantly greater activity than the N9-saline treatment in terms of all measured variables, exhibiting an apparent "biopermeation" distance approximately 3 mm further into the mucus. The DI water control treatment displayed less activity than N9-saline in terms of the vanguard penetration distance, but comparable or greater activity in terms of inhibiting kinematic variables. The saline control treatment had no effect in terms of any measured variable. Dose responses to N9 of sperm in mucus were inferred from DET results combined with direct measures of N9 diffusion. These were compared to dose responses to N9 of seminal sperm, indicating that N9 inhibits sperm motion at lower concentrations in mucus than in semen.

PIP: The double-ended test (DET) generally used to assess the ability of new spermicidal compounds to permeate cervical mucus and inhibit sperm progression overlooks the importance of sublethal effects on sperm cells. This study utilized a DET protocol that incorporated measurement of human sperm motion characteristics as an indicator of cell function within nonoxynol-9-exposed human mucus. Nonoxynol-9 was dissolved in both deionized water and saline to assess the effects of the osmolarity and pH of the delivery vehicle. All variables exhibited significant effects due to the nonoxynol-9-water treatment at distances as far as 13 mm into the mucus. The water treatment exhibited a biopermeation distance approximately 3 mm further into the mucus than the saline treatment and greater activity in terms of inhibiting kinematic variables. On the other hand, penetration of vanguard sperm was inhibited more by nonoxynol-9-saline. The reduction in straightline velocity of sperm was due more to a disruption in the pattern of motion than a reduction in overall sperm vigor. The measurements of sperm motility obtained in this study can be combined with information about local nonoxynol-9 concentrations in mucus to infer the dose-response of nonoxynol-9 against sperm in mucus. Overall, these findings indicate that the use of hypotonic solutions to deliver contraceptive agents can significantly increase the efficacy of the compounds through both increased transport rates and added bioactivity due to the carrier itself.

Measurement and modulation of nonoxynol-9 diffusion and bioactivity against spermatozoa in human cervical mucus.

Assays of sperm penetration into cervical mucus, in the configuration of double-ended tests (DETs), are an accepted format for evaluating the efficacy of sperm-directed contraceptives in mucus. In order to distinguish relative contributions of compound permeation into, and compound bioactivity within, cervical mucus with respect to vanguard sperm penetration measured in DETs, direct measurements were made of concentration profiles of the spermicide nonoxynol-9 (N9) after diffusion into mucus-filled capillary tubes. N9 was dissolved in two different delivery solutions, deionized water and saline, in an attempt to exploit a Donnan-mediated swelling of mucus for enhanced delivery of the spermicide. Average diffusion coefficients, 7 and 5 x 10(-7) cm2/sec for N9-water and N9-saline, respectively, indicate that the diffusion of N9, a surfactant material, is governed by the size of the N9 micelle rather than the molecular size, in the concentration range typically found in commercial preparations Permeation of N9 into mucus was significantly greater for water versus saline as delivery solution, although the difference was slight. A more pronounced difference between the two treatments was found in DET results, due to an osmotic and/or pH activity of the delivery solution itself against sperm in mucus.

Kinematic response of human spermatozoa to nonoxynol-9.

This study was an examination of the dose response of the kinematics of human sperm motion to 1-min and 30-min incubations with the spermicide Nonoxynol-9 (N9). At concentrations resulting in only slight reductions in percentages of motile sperm (MOT), increasing N9 decreased the progressiveness of sperm motion (reflected in decreasing straight line velocity). This decline in progressiveness resulted from both decline in the vigor (reflected in decreasing curvilinear velocity; VCL) and disruption of the pattern (reflected in decreasing linearity; LIN) of such motion. Since, after the 1-min incubation, VCL declined only slightly for seminal sperm over this range of N9 concentrations, declines in sperm progressiveness were primarily due to decreases in LIN. For sperm collected from the pellet fraction from a Percoll gradient technique, however, VCL declined substantially even at low concentrations of spermicide. These Percoll-separated sperm were, on the other hand, less sensitive to N9 than seminal sperm in terms of the dose response of MOT. This added resistance may be attributed to selection or to environmental or physiological changes caused by Percoll separation. Responses in mean amplitude of lateral head displacement (ALH) to increasing N9 also differed for the two treatments, increasing on average for seminal sperm while decreasing on average for Percoll-separated sperm.