Transdermal Dual-Controlled Delivery of Contraceptive Drugs: Formulation Development,in Vitro and in Vivo Evaluations,and Clinical Performance

Several transdermal contraceptive device (TCD) formulations were developed to provide a dual-controlled transdermal delivery of levonorgestrel (LN), a potent progestin, and 17-estradiol (E₂), a natural estrogen. Using a sensitive HPLC method, the in vitro release and skin permeation profiles of LN and E₂ from various TCD formulations were simultaneously characterized in the hydrodynamically well-calibrated Valia–Chien skin permeation cells and both were found to follow zero-order kinetics. The rates of drug release and skin permeation were observed to vary significantly depending upon some formulation parameters. Six-month stability studies were performed on seven formulations at room and elevated temperatures (37 and 45°C), and two (Formulations 4 and 5) were found to be acceptable, based on drug recovery, release rate, and skin permeation rate data. Judging from the 6-month accelerated stability studies, it is projected these two formulations will have shelf-life of at least 2 years. As a result of development of an efficient manufacturing process, Formulation 4 was selected for further evaluation. One-week primary skin irritation evaluation in 6 rabbits indicated that Formulation 4 is nonirritating, and it was thus selected for Phase I clinical bioavailability/dose proportionality studies in 12 healthy female volunteers of child-bearing age. Results of pharmacokinetic and pharmacodynamic analyses demonstrated that it is capable of achieving and maintaining a steady-state serum level of LN throughout the 3-week treatment period by weekly applications of one or two TCD patches (10 or 20 cm²). A dose proportionality was obtained in the serum drug levels, daily dose delivered, and contraception efficacy. An excellent correlation was obtained for the rates of transdermal delivery determined by the in vitro studies using human cadaver skin, the in vivo studies in rabbits, and the clinical studies in living subjects.     

Plasmid DNA vaccines: tissue distribution and effects of DNA sequence, adjuvants and delivery method on integration into host DNA

A variety of factors could affect the frequency of integration of plasmid DNA vaccines into host cellular DNA, including DNA sequences within the plasmid, the expressed gene product (antigen), the formulation, delivery method, route of administration, and the type of cells exposed to the plasmid. In this report, we examined the tissue distribution and potential integration of plasmid DNA vaccines following intramuscular administration in mice and guinea pigs. We compared needle versus Biojector (needleless jet) delivery, examined the effect of aluminum phosphate adjuvants, compared the results of different plasmid DNA vaccines, and tested a gene (the human papilloma virus E7 gene) whose protein product is known to increase integration frequency in vitro. Six weeks following intramuscular injection, the vast majority of the plasmid was detected in the muscle and skin near the injection site; lower levels of plasmid were also detected in the draining lymph nodes. At early time points (1-7 days) after injection, a low level of systemic exposure could be detected. Occasionally, plasmid was detected in gonads, but it dissipated rapidly and was extrachromosomal - indicating a low risk of germline transmission. Aluminum phosphate adjuvant had no effect on the tissue distribution and did not result in a detectable increase in integration frequency. Biojector delivery, compared with needle injection, greatly increased the uptake of plasmid (particularly in skin at the injection site), but did not result in a detectable increase in integration frequency. Finally, injection of a plasmid DNA vaccine containing the human papilloma virus type 16 E7 gene, known to increase integration in vitro, did not result in detectable integration in mice. These results suggest that the risk of integration following intramuscular injection of plasmid DNA is low under a variety of experimental conditions.     

Neurologic manifestations of human immunodeficiency virus infection in children

This report describes the neurologic manifestations of 36 children with human immunodeficiency virus (HIV) infection. In this cohort, in 16 of 21 children with acquired immunodeficiency syndrome (AIDS), three of 12 children with AIDS-related complex, and one of three asymptomatic seropositive children, a progressive encephalopathy developed. Neurologic signs were often detected early but tended to worsen coincident with progression of the immunodeficiency. The presence of progressive encephalopathy correlated with the absence of serum neutralizing antibodies to HIV and with a poor, usually fatal, outcome. The incubation period from initial HIV infection in the perinatal period to the onset of progressive encephalopathy varied from 2 months to 5 years. Intrablood-brain barrier synthesis of HIV-specific antibodies was demonstrated in eight of 14 children with AIDS and AIDS-related complex, indicating active brain infection with HIV. In three cases this was unassociated with progressive neurologic signs. Unique neuropathologic findings in children who died with HIV infection further suggest that the progressive encephalopathy is the result of primary and persistent infection of the brain with this retrovirus. These findings broaden the spectrum of HIV infection in children and have important implications for the development of antiviral therapy.     

Strain-dependent renal toxicity of a nonsteroid antiinflammatory agent

Manor Farms SPF and Charles River CD rats were given a nonsteroid antiinflammatory agent (1-p-chlorobenzylidine-5-methoxy-2-methyl-3-indeneacetic acid) po, at dose levels of 3, 6, or 12 mg/kg/day, for periods up to 29 wk. Hematuria was observed in all groups of male Manor Farms SPF rats, both control and drug-treated. The incidence of hematuria was dose-dependent, and occurred 6, 12, or 24 times more often in male rats receiving 3, 6, or 12 mg/kg/day than in the controls. In contrast, hematuria was not observed in any control females and was infrequent in drug-treated females.     

Hypolipidemic activity and toxicity studies of a styrl-hexahydroindolinol, 34–250

34–250 evoked hypocholesterolemic activity in the rat (14, 25, 31, 52, 112 mg/kg, po), dog (10, 20, 40 mg/kg, po), and monkey (30 mg/kg, po). Serum triglycerides were lowered in the rat and dog but not in the monkey. 34–250 increased [¹⁴C]acetate incorporation into liver cholesterol, but incorporation of ¹⁴C-labeled acetate into serum cholesterol was decreased. Desmosterol or 7-dehydro-cholesterol did not accumulate in serum of the three species, suggesting that inhibition of cholesterol biosynthesis by 34–250 possibly does not occur at a late stage. Normal fecal bile acid excretion was observed in rats, suggesting that cholesterol catabolism probably was not enhanced by 34–250. Compound 34–250-induced hypocholesterolemia may result from inhibition of hepatic release of this sterol into blood. The reversible hepatic lipidosis observed in rats is also possibly related to decreased hepatic transport and/or secretion of triglycerides. 34–250 did not cause a proliferation of hepatic microbodies; the lack of an increase in this fatty acid oxidizing organelle suggests that it may also have had a role in increased hepatic lipidosis. In dogs, a high incidence of severe cataracts with an early onset was induced by 20 and 40 mg/kg, po of 34–250 despite the lack of desmosterol or 7-dehydro-cholesterol in serum. The absence of these late stage intermediates of cholesterol biosynthesis in the serum of a test species does not preclude the occurrence of ocular toxicity.     

Tests for local toxicity of intramuscular drug preparations: comparison of in vivo and in vitro methods

Different concentrations of aqueous preparations of metoclopramide, gaboxadol, cis(Z)-clopenthixol, digoxin and chlorpromazine and of cis(Z)-clopenthixol acetate in Viscoleo have been tested for local toxicity after intramuscular injection in rabbits. The weight of macroscopically changed muscle tissue isolated 3 days after injection was determined. In addition, homogenized injection site muscle tissue was analysed for creatine kinase (CK) activity and by comparison with the CK activity of contralateral muscle tissue, the amount of injection site muscle tissue totally depleted for CK activity was calculated. The substances have also been tested for in vitro toxicity. The concentrations of the substances which caused 100% haemolysis of human erythrocytes or minimal cytotoxicity in cell culture assays using MRC-5 fibroblasts were estimated. Metoclopramide and gaboxadol caused no or only minimal local muscle damage in the rabbits. Metoclopramide caused local bleeding. The four other substances caused concentration-dependent muscle damage. Although there was some deviations, the two in vitro tests reflected reasonably well the in vivo findings. The results of this study suggest that the in vitro tests employed can be useful as screening tests for local toxic effect of intramuscular drug preparations. However, further studies are required before conclusion as to predictability can be drawn. The in vitro methods are inexpensive and quick, especially the haemolysis test.     

Morphologic abnormalities in potassium-deficient dogs.

Potassium deficiency was produced in 16 dogs by means of a diet containing less than 0.03% potassium. Decreases in serum potassium were first observed after 3 weeks. Morphologic changes occurred only in heart, skeletal muscle, and kidney. Focal myocardial necrosis was observed in 6 of 16 deficient dogs, and skeletal muscle degeneration and necrosis were observed in 14 of 16 deficient dogs. A complex nephropathy consisting primarily of epithelial hypertrophy and hyperplasia in the collecting tubules of the inner stripe of the outer medulla occurred in all the deficient dogs.     

Preclinical toxicological studies of carbidopa and combinations of carbidopa and levodopa

Carbidopa was administered po at 25, 45 and 135 mg/kg/day to monkeys for up to 1 year and to rats for up to 96 weeks. No physical signs were produced in monkeys although rats exhibited flaccidity at all dosage levels. No deaths, weight changes, or changes in the results of ophthalmologic, hematologic, or pathologic examinations were produced in either species. Carbidopa given to dogs resulted in pyridoxine deficiency, which was prevented by coadministration of pyridoxine. Rats and monkeys were given combinations of carbidopa and levodopa ($\text{10}\text{20}\text{,}\text{10}\text{50}\text{,}\text{and}\text{10}\text{100}\text{mg/kg/day}$). Rats given the highest dosage level daily for 2 years exhibited decreased activity, ptyalism, and retardation of weight gain. Some evidence of temporary salivary gland acinar hypertrophy was also noted. Increased activity at the middle and high dosage levels was noted in monkeys given the combined drugs for one year. No other physical signs or ophthalmologic, hematologic, or pathologic changes were noted. Carbidopa inhibits extracerebral decarboxylase activity so that lower doses of levodopa can be used in treatment of Parkinsonism with a reduction in side effects.     

Effects of chronic oral administration of a selective 5α-reductase inhibitor,finasteride, on the dog prostate

Young mature dogs received finasteride, a selective 5α-reductase inhibitor, orally at 0, 5, 15, and 45 mg/kg/day for 27 or 53 weeks. The effect of finasteride administration on prostatic size and morphology was evaluated macroscopically and microscopically. Changes in glandular and fibromuscular compartments were quantitated by a point counting method on trichrome-stained sections. Finasteride administration induced a decrease of mean prostatic weights and epithelial atrophy in all treated groups. No changes in testicular weights and morphology were observed. The greatest prostatic shrinkage was obtained in the group receiving 45 mg/kg/day for 53 weeks; compared to placebo controls, the percent decreases in absolute volumes occupied by epithelium, lumens, fibrovascular stroma, and smooth muscle were 88, 97, 51 and 72, respectively. These results clearly demonstrate that prostatic shrinkage following finasteride administration results from a decrease in both glandular and fibromuscular compartments. © 1994 Wiley-Liss, Inc.